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INTRODUCTION |
Mild oxidation of low density lipoprotein
(LDL)1 produces minimally
modified LDL (mmLDL), which is still recognized by the LDL receptor and
is not recognized by macrophage scavenger receptors (1). These
properties differentiate mmLDL from profoundly oxidized LDL (OxLDL),
which is not recognized by the LDL receptor but has enhanced uptake by
macrophage scavenger receptors, such as CD36 and SR-BI (2). However,
mmLDL possesses many pro-atherogenic properties; for example, it
induces monocyte adhesion to endothelial cells, production of colony
stimulating factors, monocyte chemotactic protein-1, and tissue factor
(1, 3, 4). In addition, it activates multiple apoptotic signaling
pathways in human coronary cells (5). Indeed, a large number of
apoptotic cells accumulate in atherosclerotic lesions (6), despite the
presence of many professional phagocytes-macrophages.
Phagocytosis of dying cells is crucial for preventing the release of
toxic cellular compounds and consequent inflammation. Inhibition of
efficient phagocytosis leads to the accumulation of proinflammatory
necrotic debris (such as oxidized lipids), plaque instability, and
thrombogenesis (7).
We have recently proposed that in mouse peritoneal macrophages
phagocytosing apoptotic cells, actin polymerization (which is necessary
for filopodia formation) depends on the activity of 12/15-lipoxygenase
(12/15-LO), which translocates from the cytosol to the cell membrane at
sites where apoptotic cells are bound (8). mmLDL carry hydroperoxides
and hydroxides of fatty acids and phospholipids, similar to those
produced by 12/15-LO catalysis (9, 10), and thereby could also
influence actin polymerization in a manner similar to that of 12/15-LO.
Because mmLDL does not bind to scavenger receptors, there are
presumably other receptors that mediate mmLDL effects.
In two recent reports, high expression of toll-like receptors (TLRs),
and especially of TLR4, has been demonstrated in macrophages in human
and mouse atherosclerotic lesions (11, 12). Moreover, the Bruneck
(Italy) Study has identified that the D299G TLR4 polymorphism that
attenuates receptor signaling is associated with a decreased risk of
atherosclerosis (13). TLR4 is a receptor playing an essential role in
innate immunity (14). It is also remarkable that the C(
260)T
polymorphism in the CD14 promoter, resulting in a significantly higher
density of CD14 on monocytes, has been identified as a risk factor for
myocardial infarction (15). In another study, a higher density of CD14
was found on monocytes from diabetic patients with cardiovascular
disease as compared with nondiabetic patients (16). CD14 does not have
its own signaling domain. Thus, upon LPS binding (assisted by
LPS-binding protein (LBP)), CD14 associates with TLR4 and triggers
TLR4-dependent signaling pathways (17). The TLR4 accessory
protein, MD-2, is also capable of binding LPS, and the TLR4/MD-2
complex can serve as an independent LPS receptor (18).
In this paper we report that mmLDL specifically binds to macrophage
CD14 and via TLR4/MD-2 activates signaling pathways leading to actin
polymerization and cell spreading. Moreover, in macrophages exposed to
mmLDL, there is a subsequent inhibition of phagocytosis of apoptotic
cells (an actin-dependent process) but, in contrast, an
enhancement of OxLDL uptake (an actin-independent pathway). These
findings provide a new link between oxidized lipoproteins and
macrophage receptors of innate immunity recognizing components of
microbial infection and also could explain in part aspects of
macrophage dysfunction that lead to the enhanced accumulation of
apoptotic cells in atherosclerotic lesions.
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EXPERIMENTAL PROCEDURES |
Cells and Reagents--
Resident peritoneal macrophages were
harvested without stimulation from 8-10-week-old female mice of
C57BL/6 (Charles River, Wilmington, MA), C3H/HeJ, or C3H/HeOuJ (Jackson
Lab, Bar Harbor, ME) strains. Murine macrophage-like cell lines J774A.1
and RAW 264.7 were from ATCC (Manassas, VA). An LPS-resistant mutant of J774, LR-9, developed by M. Nishijima, T. Nishihara, and colleagues (19) was obtained from R. Schlegel (Penn State University). These cells
have been shown to have a decreased surface expression of CD14 (19).
Studies in our lab with an anti-CD14 monoclonal antibody (R & D
Systems, Minneapolis, MN) confirmed significantly decreased CD14
expression on the surface of LR-9 cells as compared with J774 cells.
Murine fibroblast cell lines stably overexpressing human 15-LO or
-galactosidase (LacZ) were developed in our lab (9).
Chinese hamster ovary (CHO-K1) cells stably transfected with CD14 or a
CD14 mutant were generated and functionally characterized as previously
described (20). The CD14-D5 mutant had a deletion of DPRQY peptide in
the CD14 sequence. Surface expression of the protein was determined by
FACS analysis with anti-CD14 monoclonal antibody 63D3 (ATCC) (20).
Monoclonal antibody 28C5 against human CD14 was a gift from P. Tobias
(Scripps Research Institute). TLR2, TLR4, and TLR4/MD-2 stably
transfected cells were generated from Pdisplay, a CHO cell line bearing
inducible membrane CD25 under the transcriptional control of a human
E-selectin promoter containing NF-
B-binding sites, as described
before (18). TLR2 and TLR4 were engineered to express with an
N-terminal hemagglutinin tag, and MD-2 was engineered to express with a
C-terminal His tag; their surface expression was analyzed by FACS (18).
Escherichia coli LPS was purchased from List Biological Lab
(San Jose, CA). Biotinylated Re595 LPS (biotin-LPS) was a gift from R. Dziarski (Indiana University). Murine M-CSF was from R & D Systems.
LDL Isolation and Modification--
LDL was isolated from the
plasma of normolipidemic donors by sequential ultracentrifugation.
Heavily oxidized LDL (1 mg/ml) was produced by an 18-h incubation in
EDTA-free phosphate-buffered saline with 10 µM
CuSO4. Minimally modified LDL was prepared by incubating
LDL (50 µg/ml) in serum-free Dulbecco's modified Eagle's medium for
18 h with 15-LO fibroblasts (9). As previously characterized in
detail, the mmLDL generated by this procedure has normal binding to the
native LDL receptor and does not bind to scavenger receptors of
macrophages, although it is enriched by oxidized phospholipids detected
by monoclonal antibody EO6 and by lipid hydroperoxides as measured by
high pressure liquid chromatography (10, 21). Control LDL was prepared
for these experiments by an identical incubation with LacZ fibroblasts
(9) The extent of LDL oxidation was assessed by measuring its binding
to monoclonal autoantibodies EO6, specific to oxidized phospholipid or
oxidized phospholipid-protein adducts, and EO14, specific to MDA-lysine
epitopes, respectively (22).
All of the native and modified LDL preparations used in these
experiments were tested for bacterial LPS with a Limulus Amoebocyte Lysate kit (BioWhittaker, Walkersville, MD). The samples with LPS
higher than 2 ng/mg protein were discarded. Because LDL was used at a
final concentration of 50 µg/ml or less in all experiments, the LPS
contamination in experimental samples was always below 0.1 ng/ml.
F-actin Assay--
Peritoneal macrophages and J774 and
RAW cells were plated overnight in culture medium before the
experiment. Because quiescent adherent CHO cells have many stress
fibers with a high F-actin content, it is difficult to see F-actin
changes under these conditions. Thus, we plated the CHO cells for
2 h, the time sufficient for cell attachment but not for
spreading, and then stimulated them with LDL samples. The LR-9 cells,
which are larger than parental J774, were also plated for 2 h
before experiment (in this case J774, analyzed in parallel with LR-9,
were also plated for only 2 h). Relative content of F-actin in
cells was assessed by flow cytometry (8). The increase in F-actin
levels is referred to throughout the text as F-actin response. The data
were collected as histograms of distribution of cell fluorescence
intensities, and the geometrical means of each histogram were used for
further analysis. The experiments were performed in duplicate and
repeated at least three times.
CD14 Expression--
J774 and LR-9 cells were thoroughly washed,
preincubated for 2 h in serum-free medium, and then gently scraped
into bovine serum albumin/phosphate-buffered saline. The cells were
stained with biotinylated anti-mouse CD14 (R & D Systems) followed by streptavidin-Alexa Fluor 488 (Molecular Probes, Eugene, OR) and analyzed by flow cytometry.
LDL Binding to Cells--
LDL was biotinylated with EZ-Link
Sulfo-NHS-Biotin (Pierce) according to the manufacturer's protocol.
Prior to biotinylation, an aliquot of the LDL destined to be the native
LDL control, was protected from oxidation by adding 10 µM
butylated hydroxytoluene. Nonprotected biotin-LDL was further incubated
with the 15-LO cells, as described above, to produce biotin-mmLDL. The
cells were scrapped into suspension, incubated with 2-50 µg/ml of
biotin-LDL on ice for 2 h, washed, and then stained with
streptavidin-Alexa Fluor 488. The cells were also stained with
propidium iodide to differentiate live cells and analyzed on a
FACSCalibur or a FACScan. Comparison of this FACS assay with a
conventional 125I-LDL binding assay (22) showed comparable
results (data not shown).
Phagocytosis Assay--
A macrophage phagocytic population was
measured according to the procedure published in our lab (23), with
some modifications. In short, thymocytes were labeled with 1 µM of 5-chloromethylfluorescein diacetate (CellTracker
green CMFDA from Molecular Probes) followed by a treatment with
5 µM dexamethasone to induce apoptosis (23) and incubated
with macrophages. The macrophages that have engulfed CellTracker-labeled apoptotic cells differed on FACS from nonphagocytic macrophages by higher fluorescence intensity and were designated as a
phagocytic population (23).
OxLDL Uptake by Macrophages--
The cell association and
degradation of 125I-OxLDL by resident peritoneal
macrophages was determined by the method of Goldstein as performed in
our lab (22). To remove aggregates from the 125I-OxLDL
prep, it was centrifuged at 14,000 × g for 15 min and filtered through a 0.2-µm filter immediately before addition to the
cells. The amount of specific OxLDL associated or degraded per
milligram of cell protein was calculated after subtracting nonspecific
values determined in presence of excess unlabeled OxLDL.
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RESULTS |
OxLDL and mmLDL Induce Actin Polymerization and Spreading of
Macrophages--
Mouse peritoneal macrophages and the cells of the
murine macrophage-like cell line J774 exhibited a rapid and robust
increase in polymerized F-actin in response to OxLDL or mmLDL (further referred to as F-actin response) but not to native LDL, as was evident
from the increased fluorescence of the cells stained with FITC-phalloidin (Fig. 1, a and
b). Note that compared with the content of F-actin in
unstimulated peritoneal macrophages or J774 cells, OxLDL, and to an
even greater extent mmLDL, increased absolute content of F-actin by up
to 70%, whereas native LDL had no such activity. This degree of
stimulation was even greater than that observed with M-CSF (5 nM), which also increases F-actin formation (24). This gain
in the total cellular F-actin content is substantial, because actin
filaments build up the cytoskeleton and are the most abundant protein
polymer in the cell. Indeed, as might be anticipated, the increased
F-actin response was associated with a major change in cell shape, as
noted by the marked degree of cell spreading (Fig. 1, c-e).
The F-actin response to mmLDL was blunted in another macrophage line,
RAW cells; in fact, the latter did not respond to OxLDL (Fig.
1b).

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Fig. 1.
Macrophage F-actin and spreading in response
to mmLDL. Adherent cells were incubated with 50 µg/ml of
indicated LDL species, 5 nM M-CSF, or 10 ng/ml LPS for
1 h, stained for F-actin and analyzed by flow cytometry.
a, representative FACS histograms showing populations of
J774 cells incubated with native LDL (gray shading) or mmLDL
(bold line). b, geometrical means of the FACS
histograms were normalized to medium samples for each cell type.
Polymyxin B (PmB) was added at 0.5 µg/ml. *,
p < 0.01 versus media. c-e,
peritoneal macrophages were incubated with the same concentrations of
nLDL for 1 h (c), OxLDL for 1 h (d),
and OxLDL for 4 h (e), then fixed, and stained with
FITC-phalloidin, and the images were acquired. Scale bar, 15 µm.
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Concentrations of OxLDL or mmLDL between 10 and 50 µg/ml appear to
saturate the ability of OxLDL to stimulate F-actin formation (not
shown). OxLDL and mmLDL caused about a 20% increase in F-actin as
early as in 10 min (not shown), and as much as 50-70% F-actin was
attained at 1 h (Fig. 1b).
These results were not due to LPS contamination. As noted under
"Experimental Procedures," the maximum concentration of LPS in any
preparations used was below 0.1 ng/ml. Furthermore, polymyxin B did not
inhibit the mmLDL effect (Fig. 1b). When LPS was directly added at 10 ng/ml, which is at least 100-fold higher than the trace
amounts of LPS present in any of the LDL samples used, F-actin formation was increased by only 20% in J744, and in this case, polymyxin B abolished this response (Fig. 1b). Clearly, the
mmLDL effect was not produced by LPS.
Because mmLDL was produced by incubating LDL with 15-LO-overexpressing
fibroblasts (10, 21, 25), we purified it by centrifugation, 0.2-µm
filtration, and concentration on a filter with a 100-kDa cut-off. The
eluent fraction of molecular mass below 100 kDa as well as LDL
incubated with control LacZ fibroblasts (9) had a significantly lower
activity. mmLDL produced by a short period of incubation with copper (1 h), replacing 15-LO cells, was also active in eliciting the F-actin
response (data not shown).
Role of CD14 in F-actin Response--
Because the response to
mmLDL was so pronounced and mmLDL could not bind to scavenger
receptors, we focused further studies on the mechanism by which mmLDL
induced F-actin formation. We hypothesized that the differences in
amplitudes of the F-actin increase between J774 and RAW cells in
response to mmLDL and OxLDL (Fig. 1b) might be due to a
differential expression of particular receptors that bound the modified
LDL and signaled actin polymerization. We screened several receptors,
including CD14, which is known to participate in phagocytosis (26),
which binds ceramide (27), and which might be involved in LPS-induced
macrophage spreading (28). The J774 cells showed a much stronger
binding of anti-mouse CD14 and anti-mouse TLR4/MD-2 antibodies than the
RAW cells (data not shown). Further, a mutant of J774, LR-9 cells,
selected for their resistance to LPS because of the decreased surface
expression of CD14 (19) (a finding confirmed in our lab), responded to mmLDL with a significantly smaller increase in the F-actin levels compared with the parental J774 cells (Fig.
2a). The difference in F-actin
response by J774 and LR-9 cells correlated with nearly 6-fold increased
specific binding of mmLDL to J774 but not to LR-9 cells (Fig.
2b). (Note that the binding of biotinylated mmLDL to
J774 cells was abolished by a 25-fold excess of unlabeled mmLDL). Again, note that polymyxin B had no effect on the binding of mmLDL to
J774 cells (Fig. 2b).

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Fig. 2.
F-actin response and mmLDL binding to J774
and LR-9 cells. a, flow cytometry analysis of F-actin
levels in J774 and CD14-deficient LR-9 cells exposed for 1 h to 50 µg/ml of nLDL or mmLDL. The data are shown as percentages of F-actin
change compared with the cells in media without any LDL addition.
b, flow cytometry analysis of biotinylated LDL binding to
J774 and LR-9. The geometrical means of FACS histograms (minus
"no LDL" controls) are shown. Some samples contained a 25-fold
excess of nonlabeled LDL or 0.5 µg/ml polymyxin B
(PmB).
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The experiments above provided substantial support for specific binding
of mmLDL to CD14. To provide direct evidence, we next tested whether
overexpression of CD14 would lead to an increased F-actin response to
mmLDL. A CHO cell line stably transfected with human CD14 (CD14 cells)
responded to mmLDL with significantly higher levels of F-actin than the
wild type CHO-K1 cells (Fig. 3a). Although changes in
F-actin levels in CD14 cells were not as pronounced as in macrophages,
the 25% increase is still a substantial change in F-actin formation
for these large ovary cells, which are not professional phagocytes and
do not rapidly form filopodia, as do macrophages. Accordingly, binding
of biotinylated mmLDL to the CD14 cells was significantly higher than
to the wild type CHO cells (Fig. 3, b and c). To
demonstrate specific binding to the CD14 cells, biotinylated mmLDL
binding curves were determined in the absence or presence of unlabeled
mmLDL. As shown in Fig. 3b, there was specific and saturable
binding of mmLDL with a calculated Kd of 15 ± 3 µM. The specific binding of mmLDL to CD14 cells was
10-fold higher than to control CHO cells (Fig. 3c). The
mmLDL binding correlated with increased binding of LPS to the CD14
cells (Fig. 3e). This increased LPS binding was specific for
CD14, because monoclonal antibody 28C5, directed against the LPS
binding site of CD14, reduced LPS binding to levels seen in nontransfected CHO cells (Fig. 3e and Ref 20). However,
antibody 28C5 did not block either the F-actin response to mmLDL or
mmLDL binding to the CD14 cells (Fig. 3, a and
d). These data suggest that although mmLDL has specific
binding to CD14, it binds at a site distinct from the LPS binding site
(at least that domain to which 28C5 binds). Further support for this
comes from studies of binding to the CD14 mutant CD14-D5, in which the
peptide DPRQY is deleted, rendering CD14 unable to support binding of
LPS (20). Transfection of CHO cells with CD14-D5 resulted in CHO cells
in which no specific LPS binding was observed (Fig. 3e).
However, binding of mmLDL to CD14-D5 was inhibited by only 25%
compared with CD14 cells (Fig. 3d), and the ability to
stimulate F-actin formation was not significantly decreased (Fig.
3a). These data confirm that mmLDL binds to CD14 but to a
region distinct from the LPS-binding site.

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Fig. 3.
F-actin response and mmLDL binding to
CD14-transfected CHO cells. a, flow cytometry analysis
of F-actin levels in wild type or CD14-transfected CHO cell lines
exposed for 1 h to 50 µg/ml of nLDL or mmLDL. The data are shown
as percentages of F-actin change compared with the cells in medium
without any LDL addition. b and c, binding curve
of biotinylated LDL binding to CD14 (b) and nontransfected
CHO (c) cells. Geometrical means of FACS histograms (minus
"no LDL" controls) are shown. Total binding designates biotin-LDL
binding in the absence of a competitor. Nonspecific binding is binding
in the presence of a 20-fold excess of nonlabeled LDL. Specific binding
is a result of subtraction of nonspecific from total binding.
d, specific binding of nLDL and mmLDL to CD14,
CD14-D5, and CHO cells in the presence and absence of anti-CD14
antibody 28C5 (6 µg/106 cells), added 1 h prior to
addition of LDL. *, p < 0.05 versus CHO-K1.
e, biotinylated LPS (50 ng/106 cells) binding to
CD14, CD14-D5, and CHO cells in the presence and absence of anti-CD14
antibody 28C5 (6 µg/106 cells).
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Role of TLR4/MD-2 in F-actin Response--
Because most
CD14-dependent LPS effects are believed to require the TLR4
signaling pathways (29), we studied whether the F-actin response to
mmLDL depended on TLR4. Peritoneal macrophages from C3H/HeJ mice have
normal CD14 expression, but they are resistant to LPS because of a
mutation in the TLR4 gene, Tlr4Lps-d (30). In
contrast, macrophages from C3H/HeOuJ mice carry a wild type
Tlr4Lps-n and are sensitive to endotoxin. The
F-actin response to mmLDL was significantly blunted in
Tlr4Lps-d macrophages compared with
Tlr4Lps-n macrophages (Fig.
4a). Moreover, in agreement
with our experiments with C57BL/6 macrophages (Fig. 1,
c-e), Tlr4Lps-n macrophages from
C3H/HeOuJ mice readily spread following a 1-h exposure to mmLDL (Fig.
4, b and c). In contrast, the morphology of the
Tlr4Lps-d macrophages from C3H/HeJ mice exposed
for the same time to mmLDL did not significantly differ from those
exposed to nLDL or medium alone (Fig. 4, d and
e).

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Fig. 4.
Role of TLR4/MD-2 in F-actin response to
mmLDL. Peritoneal macrophages were harvested from C3H/HeOuJ
(TlrLps-n) and C3H/HeJ
(TlrLps-d) mice, plated, and then incubated for
1 h with 50 µg/ml of nLDL or mmLDL. a, F-actin levels
were measured by flow cytometry. b-e, phase contrast
microphotographs of the macrophages at the end of incubation:
TlrLps-n + nLDL (b),
TlrLps-n + mmLDL (c),
TlrLps-d + nLDL (d), and
TlrLps-d + mmLDL (e). f,
flow cytometry analysis of F-actin levels in indicated CHO cell lines.
TLR2, TLR4, and TLR4/MD-2 stably transfected cells were generated from
the CHO cell line Pdisplay (18).
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To support the hypothesis of TLR4 involvement in mediating the mmLDL
induction of actin polymerization, we next compared the F-actin
response of CHO cells stably transfected with either TLR2, TLR4, or
TLR4/MD-2. Of these, only the TLR4/MD-2 cells responded to mmLDL with
significantly increased levels of F-actin as compared with the control
CHO cells (Fig. 4f). Thus, mmLDL can directly activate
TLR4/MD-2 in these cells when these molecules are overexpressed.
Next, we tested whether inhibition of several key components of known
TLR signaling pathways (14) will influence the F-actin response. The
phosphoinositide 3-kinase (PI3K)-specific inhibitors wortmannin and
LY294002 (31) efficiently inhibited actin polymerization in J774 cells
(Fig. 5). The effect of wortmannin was
dose-dependent in the range of 10-100 nM (not
shown). In contrast to the PI3K inhibitors, the inhibitor of NF-
B
translocation, SN50, and the p38 MAPK inhibitor, SB202190, did not have
any significant effect (Fig. 5). Wortmannin is among the most selective
kinase inhibitors available (32), and it inhibited mmLDL-induced
F-actin formation (Fig. 5) at its reported IC50
concentrations. SB202190 was inactive even at a concentration that was
100-fold higher than its reported IC50 (32). SN50 was used
at the concentration shown to be effective for NF-
B inhibition in
many macrophage cell types (33, 34). We also tested the effects of PI3K
inhibitors in the CHO cells transfected with CD14 and TLR4/MD-2.
Wortmannin and LY294002 inhibited spreading of CD14 and TLR4/MD-2 CHO
cells, but the absolute reduction in F-actin formation was not
statistically significant (data not shown), presumably because of the
low amplitudes of F-actin response in transfected CHO cells (as
discussed above).

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Fig. 5.
Effect of specific signaling inhibitors on
F-actin response. J774 cells were preincubated for 30 min with
either medium alone (control), 50 nM wortmannin
(wortm), 10 µM LY294002, 50 µg/ml SN50, or 5 µM SB202190 and then incubated for 1 h with medium
or 50 µg/ml mmLDL. F-actin levels were measured by FACS. The numbers
were normalized to control (no inhibitor treatment) samples. *,
p < 0.001 versus control. FITC,
fluorescein isothiocyanate
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Effect of mmLDL-induced Actin Polymerization on Phagocytosis of
Apoptotic Cells and Uptake of OxLDL by Macrophages--
We originally
began these studies because of our interest in the mechanism by which
OxLDL affected macrophage uptake of apoptotic cells (8, 23). Therefore
we examined what impact the mmLDL-induced F-actin formation in
macrophages would have on the subsequent ability of the macrophages to
phagocytose apoptotic cells. The use of mmLDL in this case helps avoid
any possible competition between the OxLDL added during the
preincubation and the apoptotic cells used to test phagocytosis,
because both bind to scavenger receptors (23). Preincubation with mmLDL
resulted in a markedly decreased ability of macrophages to subsequently
phagocytose apoptotic cells. In four separate experiments, mmLDL
decreased the uptake of apoptotic cells by 35-60% (Fig.
6a). To explain this, we
hypothesized that the mmLDL-induced actin polymerization caused
extensive cell spreading, which in turn led to a relative inability to
mobilize actin polymerization sufficient for phagocytosis. To test this hypothesis, we pretreated the cells with M-CSF, which also caused a
comparable increase in F-actin (Fig. 1b) but in an
mmLDL-independent manner. M-CSF pretreatment also resulted in
subsequent inhibition of phagocytosis of apoptotic cells similar to
that seen with mmLDL (Fig. 6a).

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Fig. 6.
Effect of preincubation with mmLDL on
macrophage phagocytic function and OxLDL uptake. Resident
macrophages were preincubated for 4 h with medium only, 50 µg/ml
nLDL or mmLDL, or 5 nM M-CSF and then exposed to
labeled apoptotic thymocytes (a) or 125I-OxLDL
(b). Macrophage phagocytic populations (a) and
specific 125I-OxLDL association and degradation by
macrophages (b) were measured. *, p < 0.01;
**, p < 0.05 versus medium.
c-e, phase contrast photographs of macrophage cultures
incubated for 4 h with nLDL (c), mmLDL (d),
or M-CSF (e).
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Uptake of monomeric OxLDL requires the presence of functional scavenger
receptors but does not involve an actin-dependent phagocytic mechanism (35). In contrast, uptake of apoptotic cells
occurs in part via scavenger receptors and is
actin-dependent. To demonstrate that nonphagocytic
scavenger receptor-mediated uptake was not influenced, we repeated the
mmLDL preincubation with macrophages, but instead of examining the
uptake of apoptotic cells, we examined the uptake of aggregate-free
125I-OxLDL. In contrast to inhibition of phagocytosis,
pretreatment of macrophages with mmLDL or M-CSF significantly increased
subsequent specific uptake and degradation of 125I-OxLDL
(Fig. 6b). This result proves that the mmLDL mediated inhibition of phagocytosis of apoptotic cells is not due to
down-regulation of scavenger receptors and, to the contrary, is
consistent with the known ability of OxLDL (and M-CSF) to up-regulate
CD36 (36, 37). Morphologically, macrophages treated with both mmLDL and M-CSF, but not with native LDL, were found to be extensively spread (Fig. 6, c-e), consistent with the hypothesis that the
shape and/or actin-related changes in macrophages were the cause of
inhibited phagocytosis of apoptotic cells.
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DISCUSSION |
This study reports several novel observations. We demonstrate that
OxLDL and mmLDL stimulate fast and robust actin polymerization and
macrophage spreading (Fig. 1). Because mmLDL does not bind to scavenger
receptors (1), it was unclear how the mmLDL affected F-actin formation.
Here we show that mmLDL specifically binds to CD14 (Figs. 2 and 3) and
initiates F-actin formation through a signaling pathway that depends on
TLR4/MD-2 (Fig. 4) and PI3K (Fig. 5). The mmLDL-induced macrophage
spreading culminates on the one hand in a reduced ability of
macrophages to efficiently phagocytose apoptotic cells but, on the
other hand, in the enhanced uptake of OxLDL, two consequences that
would be predicted to enhance atherogenesis (Fig. 6).
Our data demonstrate that mmLDL and OxLDL produced comparable increases
in macrophage F-actin (Fig. 1b). In accordance with our
data, actin polymerization was recently reported to accompany OxLDL-mediated monocyte adhesion and transendothelial migration (38).
We used a well characterized technique for producing mmLDL by
incubating LDL with 15-LO-overexpressing cells, which seed fatty acid
hydroperoxides into LDL (9, 10, 21). As previously demonstrated, this
mmLDL still binds to the LDL receptor and, although enriched in
hydroperoxides and oxidized phospholipids, nevertheless does not bind
to scavenger receptors that bind OxLDL (9). Thus, we asked whether the
mmLDL effect on actin cytoskeleton occurs through binding to cell
surface receptors other than OxLDL scavenger receptors. Native LDL did
not stimulate F-actin formation (Figs. 1-3). Preliminary studies with
J774 and LR-9 cells suggested the involvement of CD14 (Fig. 2). Indeed,
we found that specific binding of biotin-mmLDL was 10-fold higher to
CD14 cells than to control CHO cells, and this corresponded to
increased F-actin formation by the CD14 cells in response to mmLDL
(Fig. 3). Because we performed our experiments in medium without serum,
this implies that LBP is not obligatory for the mmLDL binding to CD14.
In the case of LPS, LBP basically catalyzes transfer of monomers from LPS aggregates and presents the LPS monomers to CD14. In the case of
mmLDL, oxidation changes phospholipid presentation on the LDL particle
(39), and this alone, without LBP catalysis, could be a sufficient
transition causing mmLDL to be recognized by CD14. In favor of this
hypothesis are data showing binding of isolated phospholipids to both
membrane and soluble CD14 (40, 41). On the other hand, oxidation of the
fatty acid chains of phospholipids in the LDL particle changes
presentation of the phospholipid head groups, making them immunogenic
(39). Similar changes occurring in mmLDL may lead to CD14 recognition.
The anti-CD14 antibody 28C5 or the DPRQY deletion in the CD14 sequence
blocked the binding of LPS to CD14, but it did not block mmLDL binding
(Fig. 3, d and e), suggesting that CD14-binding sites for LPS and mmLDL differ. Interestingly, binding of
phosphatidylinositides to CD14 has been shown to be blocked by
anti-CD14 antibodies and to compete with LPS binding to CD14 (40),
suggesting similar CD14 binding sites for phosphatidylinositides and
LPS. Future studies using various CD14 mutants and monoclonal
antibodies to different CD14 epitopes will help map the mmLDL-binding
site on CD14. In a paper published after completion of this work,
Bochkov et al. (42) demonstrated that a model oxidized
phospholipid, 1-palmitoyl-2-(5-oxovaleroyl) phosphatidylcholine,
competed with the ability of LPS to bind to CD14 and to activate the
NF-
B pathway. Whether this is occurring via a similar mechanism as
described here is uncertain, because our studies suggest that the mmLDL site on CD14 is different from that of LPS.
The TLR4/MD-2-CHO cells also responded to mmLDL with significantly
elevated F-actin (Fig. 4). Although TLR4 alone does not bind LPS, MD-2
has been shown to bind LPS without assistance from either LBP or CD14,
and the LPS-MD-2 binding constant is comparable with that of LPS-CD14
(18). It could be also that mmLDL binds to MD-2, although it was not a
focus of this study. In several studies, TLR4 activation by various
agonists has been reported, with indirect data suggesting potential
binding of these agonists (43, 44). Studies with isolated MD-2 and CD14
molecules will help elucidate their binding to mmLDL. Our other
experiments also support the role of TLR4 in mediating the F-actin
response to mmLDL. C3H/HeJ macrophages with the mutated Tlr4
gene lost their ability to respond to mmLDL by either enhanced actin
polymerization or spreading (Fig. 4). The C3H/HeJ mice are known not
only for their resistance to LPS but also for resistance to
cholesterol-induced atherosclerosis (45, 46). Because we show that the
mmLDL-induced macrophage spreading inhibits phagocytosis of
apoptotic cells (Fig. 6) and could be therefore considered
pro-atherogenic, it is tempting to propose that among other
factors (46), the TLR4Lps-d mutation and mmLDL
resistance make the C3H/HeJ mice less susceptible to atherosclerosis.
However, C57BL/10ScN mice, in which the Tlr4 gene is deleted
(Tlr4lps-del), cross-bred with
ApoE
/
mice, did not show any changes in atherosclerosis
progression compared with control ApoE
/
mice when these
mice were fed a Western diet (47). Such a diet, which induces extreme
hypercholesterolemia and excessive atherogenic pressure, may have
obscured an ability of TLR4 to modulate atherogenesis. Similarly, Rag
(1 or 2)-deficient mice bred into the ApoE
/
background
do not show any attenuation in lesion formation when such mice are fed
a Western diet, but, if chow-fed, reductions of atherosclerosis of
40-80% are found (48, 49). In addition, importance of this pathway in
humans was strongly suggested by the recent Brunek (Italy) study, which
identified that the D299G TLR4 polymorphism, attenuating receptor
signaling, was associated with a decreased risk of atherosclerosis
(13).
TLR signaling in most cases is associated with pro-inflammatory gene
expression (14). What is known about the role of TLR in the cell
cytoskeleton regulation? TLR2 activates NF-
B via PI3K and a Rho
family small GTPase Rac1 (50). In addition to transcriptional
regulation, PI3K and Rac are well known regulators of actin
polymerization and macrophage phagocytosis (51, 52). Our data show that
PI3K might also be involved in mmLDL-induced actin polymerization (Fig.
5), although it is not yet clear whether the PI3K activation depends on
TLR4 signaling in this case. Another study reports that a high dose of
LPS stimulates spreading of J774 cells via activation of
2-integrin (28). The authors of this elegant work show
that the LPS-induced spreading (occurring in 10 min) is linearly
dependent on the signaling chain, which is common for TLRs: from MyD88
to IRAK to p38 MAPK. Subsequently, p38 controls spreading independently
of its role in transcription but rather through activation of another
small GTPase of the Ras family, Rap1, which in turn activates
2-integrin (28). We also found that an inhibitor of
NF-
B nuclear translocation did not affect the F-actin response to
mmLDL (Fig. 5). However, unlike in the studies of Schmidt et
al. (28), p38 inhibition did not significantly influence the mmLDL
effect (Fig. 5).
Our finding that the biological effect of mmLDL is mediated by the LPS
receptors agrees with a general concept of these receptors' function.
Both CD14 and TLR family receptors are "pattern recognition receptors" activated by a variety of chemically different ligands. Besides LPS, these receptors recognize conserved products of microbial metabolism, such as peptidoglycan, lipoteichoic acids, and other components of bacterial cell walls (53, 54), as well as nonmicrobial substances. For instance, such different stimuli as the plant product
Taxol (43), heat shock protein 60 (55), and the respiratory syncyntial
virus coat protein F (44) have all been reported as TLR4 agonists. CD14
binds ceramide, which can be found in modified LDL (27, 56). The
results of our study suggest that structural changes in the LDL
particle upon its mild oxidation make it also recognizable by immune
competent cells and lead to TLR4 activation. Further studies will be
needed to define the ligand(s) on mmLDL mediating the binding and
biological effects.
These data and other results from our group support the hypothesis of
convergence of immune responses to oxidation-specific self-antigens and
to microbial infection. This hypothesis was first substantiated by data
from our group showing that a natural autoantibody to mmLDL, EO6, is
genetically identical to the classic germline anti-phosphorylcholine
antibody T15, which binds avidly to Streptococcus
pneumoniae. More specifically, EO6/T15 recognize bacterial cell
wall polysaccharides that are rich in phosphorylcholine (57), as well
as phosphorylcholine-containing oxidized phospholipids present in
oxidized LDL (22) and cells undergoing apoptosis (23). The present
study demonstrates correspondence not only in humoral (EO6-T15
antibody) but also in cellular (CD14 and TLR4 receptors) immune
responses to mmLDL and to microbial infection.
Finally, this study shows that the CD14/TLR4/MD-2-mediated macrophage
spreading in response to mmLDL inhibits phagocytosis of apoptotic cells
(Fig. 6a). This result cannot be explained by the
competition between mmLDL and apoptotic cells for the same macrophage
receptors because mmLDL does not bind to scavenger receptors (1, 21).
Moreover, mmLDL pretreatment actually increases the subsequent uptake
of OxLDL by macrophages (Fig. 6b), showing that scavenger
receptor expression was not suppressed and in fact was increased. In a
similar manner, macrophage spreading caused by M-CSF also resulted in
decreased phagocytosis, but increased OxLDL uptake (Fig. 6,
a and b). The exact mechanisms by which macrophages exposed to mmLDL (or M-CSF) become less efficient phagocytes but have a higher rate of OxLDL uptake are not yet known.
Presumably, the higher rate of OxLDL uptake induced by mmLDL exposure
is mediated by activation of peroxisome proliferator-activated receptor
and enhanced CD36 expression (36). Because mmLDL, as well
as OxLDL, is prevalent in atherosclerotic lesions, our observations
could have important implications in understanding macrophage function
in atherosclerotic lesions. Considering phagocytosis of apoptotic
cells as an anti-inflammatory process and extensive uptake of OxLDL as
a pro-atherogenic event, one would predict a net pro-atherogenic role
of mmLDL as a consequence of these effects on macrophages. Thus, this
study defines novel pathways by which both mmLDL and microbial
infection could influence the progress of atherogenesis.