© 1998 American Heart Association, Inc.
Original Contributions |
Chemokine Receptor CCR2 Expression and Monocyte Chemoattractant Protein-1–Mediated Chemotaxis in Human Monocytes
A Regulatory Role for Plasma LDL
From the Department of Medicine, University of California at San Diego, La Jolla, Calif. R.K.T. is presently at the Department of Medicine/Cardiology, University of Pennsylvania Medical Center, Philadelphia.
Correspondence to Oswald Quehenberger, University of California San Diego, Department of Medicine 0682, 9500 Gilman Dr, La Jolla, CA 92093-0682. E-mail oquehenberger{at}UCSD.edu
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Abstract—The subendothelial accumulation of macrophage-derived foam cells is one of the hallmarks of atherosclerosis. The recruitment of monocytes to the intima requires the interaction of locally produced chemokines with specific cell surface receptors, including the receptor (CCR2) for monocyte chemoattractant protein-1 (MCP-1). We have previously reported that monocyte CCR2 gene expression and function are effectively downregulated by proinflammatory cytokines. In this study we identified low density lipoprotein (LDL) as a positive regulator of CCR2 expression. Monocyte CCR2 expression was dramatically increased in hypercholesterolemic patients compared with normocholesterolemic controls. Similarly, incubation of human THP-1 monocytes with LDL induced a rapid increase in CCR2 mRNA and protein. By 24 hours the number of cell surface receptors was doubled, causing a 3-fold increase in the chemotactic response to MCP-1. The increase in CCR2 expression and chemotaxis was promoted by native LDL but not by oxidized LDL. Oxidized LDL rapidly downregulated CCR2 expression, whereas reductively methylated LDL, which does not bind to the LDL receptor, had only modest effects on CCR2 expression. A neutralizing anti–LDL receptor antibody prevented the effect of LDL, suggesting that binding and internalization of LDL were essential for CCR2 upregulation. The induction of CCR2 expression appeared to be mediated by LDL-derived cholesterol, because cells treated with free cholesterol also showed increased CCR2 expression. These data suggest that elevated plasma LDL levels in conditions such as hypercholesterolemia enhance monocyte CCR2 expression and chemotactic response and potentially contribute to increased monocyte recruitment to the vessel wall in chronic inflammation and atherogenesis.
Key Words: CCR2 • monocyte chemoattractant protein-1 • LDL • chemotaxis
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Introduction |
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The first grossly visible lesion in human and experimental atherosclerosis is the fatty streak, composed of cholesterol-loaded foam cells. Most of these cells are derived from circulating monocytes that have taken up residence in the subendothelial space.1 2 3 4 Increased monocyte recruitment can be explained in part by the production of chemoattractant factors and adhesion molecules by the cells of the arterial wall. LDL that has undergone minimally oxidative modifications has been shown to induce in aortic endothelial and smooth muscle cells the secretion of monocyte chemoattractant protein-1 (MCP-1),5 6 a member of the family of C-C or ß-chemokines that attract mainly monocytes.7 8 9 Additional factors that can augment the recruitment of monocytes, primarily through induction of MCP-1 secretion by vascular smooth muscle and endothelial cells, include thrombin, which is generated at sites of vascular injury, macrophage colony–stimulating factor, and other inflammatory cytokines present in atherosclerotic lesions.10 11 12 13 Atherosclerotic lesions have a predilection for areas where the blood flow is disturbed, and hemodynamic forces may play a role in monocyte recruitment. Activation of endothelial cells by exposure to fluid mechanical forces mediates the secretion of MCP-1 and the differential expression of adhesion molecules implicated in atherosclerosis.14 15 16 The strong expression of MCP-1 in human and rabbit atherosclerotic lesions is a further indication that this chemokine might be importantly involved in monocyte recruitment during atherogenesis.17 18
Monocyte recruitment is a multistep process.19 The firm adhesion to the endothelium is supported by adhesion molecules, and subsequent transendothelial migration is mediated by the interaction of locally produced chemokines with cell surface receptors.9 20 The receptor for MCP-1, CCR2, has been cloned recently and belongs to the family of 7 transmembrane-spanning, GTP-binding protein–coupled receptors.21 Two isoforms of the human MCP-1 receptor have been identified. Both CCR2A and CCR2B, the latter being the predominant form on human monocytes, are highly homologous and differ only in their carboxyl tail.22 Recent studies in our laboratory have demonstrated that monocyte CCR2 expression is affected by inflammatory modulators and is effectively downregulated by proinflammatory cytokines, growth factors such as granulocyte-macrophage colony–stimulating factor, macrophage colony–stimulating factor, and MCP-1 itself.23 Thus, factors produced by cells of the vessel wall can control monocyte responses through modulation of CCR2 expression, may prevent a disproportionate inflammatory response, and support the arrest of recruited monocytes in lesions.
In addition to cytokines and modified forms of LDL, native LDL at concentrations typical for hypercholesterolemia has been demonstrated to affect endothelial gene expression, resulting in increased monocyte binding.24 Reciprocally, monocytes from hyperlipidemic subjects or animal models have been shown to be more responsive to inflammatory stimuli and have shown increased cell motility, chemotaxis, and adherence to cultured endothelial cells compared with monocytes from normolipidemic controls.25 26 27 28 However, the mechanisms by which these functional changes occur are not known.
In the present study, we examined the effects of atherogenic lipoproteins on CCR2 expression. Our data demonstrate that native plasma LDL, but not oxidized LDL, rapidly upregulates CCR2 expression and profoundly enhances the monocyte chemotactic response to MCP-1. These results indicate that elevated plasma LDL levels in hypercholesterolemia may increase monocyte chemotactic responses and cause excessive monocyte infiltration and accumulation in atherosclerotic lesions.
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Methods |
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Materials
Cell culture medium and Hanks' balanced salt solution were purchased from Life Technologies, Inc. FCS was from Hyclone Laboratories. Recombinant human MCP-1 and the neutralizing monoclonal anti–human MCP-1 antibody were obtained from R&D Systems Inc. 125I–MCP-1 (specific activity, 2200 Ci/mmol) was from DuPont-NEN. The chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine and cholesterol were purchased from Sigma. Dibutyl phthalate and dioctyl phthalate were from Aldrich. The anti-LDL receptor IgG C7 was obtained from American Type Culture Collection, Manassas, Va. All other reagents were of the highest purity available.
Cell Culture and Incubation Conditions
THP-1 monocytes (American Type Culture Collection) were
maintained in RPMI 1640 medium supplemented with 10% FCS, 100 U/mL
penicillin, 100 µg/mL streptomycin (Irvine Scientific), and 20
µmol/L mercaptoethanol in a humidified 5% CO2
atmosphere at 37oC. The cell density was kept
below 0.5x106 cells/mL. For isolation of human
monocytes, whole blood containing 3.0 mmol/L EDTA was
centrifuged at 1500g for 30 minutes at
4oC, and the buffy coat with adjacent plasma
was collected. The cell suspension (7 mL) was layered onto 3 mL of
Histopaque 1.077 (Sigma) and separated by
centrifugation at 600g for 30 minutes at
22oC. The mononuclear cells were washed with
PBS containing 0.1% BSA and 3.0 mmol/L EDTA and plated in 10%
autologous serum for 2 hours at 37oC in a 5%
CO2 atmosphere, and the adherent monocytes were
harvested.
Incubations with native LDL, various forms of oxidized LDL, or methylated LDL were conducted in the presence of 10% serum to avoid any cell-mediated oxidation of LDL. The anti–LDL receptor IgG included in some of the experiments was used at a 1:200 dilution. For longer incubations the antibody was replenished every 24 hours. Direct loading of THP-1 monocytes with cholesterol was achieved by incubating the cells with 25 µg/mL of free cholesterol for 24 hours. The ethanol used to dissolve cholesterol did not affect cell viability, which was >95%, as determined by trypan blue exclusion. After incubation under various conditions, the cells were washed 3 times with PBS and used for the analyses as described. Care was taken to not contaminate the incubation medium with endotoxin, and its concentration never exceeded 0.03 ng/mL, which was determined by the timed gel-formation method as described by the supplier of the endotoxin kit (Sigma).
Preparation of Lipoproteins
Native human LDL (d=1.03 to 1.63 g/mL) was isolated
by ultracentrifugation29 and
stored at 4oC in PBS containing 0.3 mmol/L
EDTA. To prevent oxidation, BHT was added,30 and
the LDL preparations contained 
0.3 nmol of thiobarbituric
acid–reactive substances (TBARS) per mg protein, as determined by
fluorometric assay.31 Mildly oxidized LDL was
prepared by incubating 2 mg/mL of LDL not protected by antioxidants
with 2 µmol/L CuSO4 in EDTA-free PBS for 4
hours at 37oC as
described.32 Fully oxidized LDL was prepared by
incubation of freshly isolated LDL (100 µg/mL) in Ham's F-10 medium
with 10 µmol/L CuSO4 for 24 hours as
described.33 The extent of oxidation was
determined, and mildly oxidized LDL contained 4 nmol TBARS/mg
protein and displayed a relative electrophoretic mobility of 1.2
(native LDL, 1.0) by agarose gel electrophoresis at 100 V for 30
minutes. Fully oxidized LDL contained, on average, 55 nmol TBARS/mg
protein and displayed a relative electrophoretic mobility of 3.3.
Methylated LDL was prepared by reductive methylation as
described.34 In brief, 1 mg
NaBH4 was added to LDL (5 mg of LDL protein per
mL) in 1 mL of 0.15 mmol/L NaCl and 0.3 mmol/L EDTA, pH 7.0,
at 4oC. Immediately thereafter, 2 µL of 37%
formaldehyde was added, and the addition of formaldehyde was repeated 4
times at 5-minute intervals. The entire sequence was repeated once to
achieve more extensive methylation. After dialysis against PBS, the
extent of lysine modification was estimated by the
trinitrobenzenesulfonic acid assay.35 This
procedure modified 80% of the LDL lysine residues, and the methylated
LDL was not recognized by the LDL receptor, which was tested in
competition experiments using
125I-LDL.36 All lipoprotein
samples were dialyzed against PBS (pH 7.4) and sterile-filtered before
use in cell culture experiments. The endotoxin level of the LDL stock
solutions never exceeded 0.1 ng/mg LDL protein, and the level in the
incubation medium at the highest concentrations of native, mildly
oxidized, and fully oxidized LDL used in the experiments never exceeded
0.05 ng/mL. At this concentration endotoxin does not affect CCR2
expression.37
Determination of Cell Surface CCR2 and Equilibrium Binding
Analysis
THP-1 monocytes incubated under various conditions were
isolated by centrifugation and washed 3 times with PBS,
and cell number was determined by counting an aliquot, stained with
crystal violet, under a microscope. The cells were resuspended at
2x107 cells/mL in 200 µL of binding buffer
consisting of 50 mmol/L HEPES, 1 mmol/L
CaCl2, 5 mmol/L MgCl2,
and 0.5% BSA, pH 7.2, and binding assays were carried out as
described.23 In brief, the cells were incubated
for 90 minutes at 25oC with 0.07 nmol/L
125I–MCP-1 (specific activity, 2200 Ci/mmol) in
the absence or presence of 100 nmol/L unlabeled MCP-1. The reaction was
terminated by separating the cells from the incubation buffer by
centrifugation through a 1:1 mixture (vol/vol) of
dibutyl phthalate and dioctyl phthalate, and the radioactivity
associated with the cell pellet was counted. The specific
125I–MCP-1 binding was determined by subtracting
the nonspecific binding estimated in the presence of 100 nmol/L
unlabeled MCP-1 from total binding. To determine the exact binding
affinity as given in Table 2
and validate the estimation of
Bmax by the end-point method described above, the
cells were incubated with 0.07 nmol/L 125I–MCP-1
in the presence of increasing concentrations (from 0.001 to 100 nmol/L)
of unlabeled MCP-1, and the data were analyzed using the
LIGAND program.38 The receptor number
determined by ligand binding was normalized to cell number and
expressed as fmol/106 cells.
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Analysis of CCR2 Transcripts
Total RNA was isolated by guanidinium
thiocyanate–phenol-chloroform extraction.39 The
mRNA was reverse-transcribed with Superscript II (Life Technologies
Inc) using 2 µg of total RNA and 0.12 nmol
oligo(dT)10 primers (Boehringer
Mannheim). CCR2 gene expression was then estimated by semiquantitative
polymerase chain reaction (PCR). Two primers with the sequences
5'-ATGCTGTCCACATCTCGTTCTCG (sense primer) and
5'-TTATAAACCAGCCGAGACTTCCTGC (antisense primer) were used to amplify
the full-length cDNA of CCR2B (1083 bp) as described
previously.23 Routinely, 25 to 30 PCR
amplification cycles were used, and the concentrations of the
reverse-transcribed template cDNA in the reaction mixture was adjusted
to obtain a linear relation between template and product. The
specificity of the amplification was confirmed by DNA sequencing of the
PCR product. The amplified DNA was analyzed by agarose gel
electrophoresis and stained with ethidium bromide. The intensity of the
stained band was analyzed by densitometric scanning (Image
Quant, Molecular Dynamics) and compared with that of the internal
standard. As an internal standard, GAPDH was amplified and
analyzed under identical conditions using
5'-TCGGAGTCAACGGATTTGGTCGTA as the sense primer and
5'-ATGGACTGTGGTCATGAGTCCTTC as the antisense
primer.40 The expected size of the amplified cDNA
was 520 bp, which was used to normalize relative changes of CCR2 mRNA.
As a negative control to test for contamination with genomic DNA, RNA
samples without prior treatment with reverse transcriptase were used
for PCR analysis using the primers described above. All samples
tested negative for genomic DNA contamination.
Chemotaxis Assay
THP-1 monocytes incubated under various conditions were rinsed 3
times with PBS and then suspended at a concentration of
1.5x106 cells/mL in chemotaxis buffer consisting
of Tyrode's salt buffer (Sigma), 1% NaHCO3, and
0.5% BSA, pH 7.4. The chemoattractants MCP-1 or
N-formyl-methionyl-leucyl-phenylalanine were added to the
lower chamber of the 48-well microchemotaxis Boyden chamber
(Neuroprobe) at a concentration of 10 nmol/L in chemotaxis buffer. The
cell suspension (51 µL) was added to the upper chamber, which was
separated from the lower chamber by a 5-µm-pore-size polycarbonate
membrane (Poretics). After incubation for 1 hour at
37oC in a 5% CO2
atmosphere, the side of the polycarbonate membrane in contact with the
cell suspension was scraped and washed to remove any cells. After
fixation in 1% glutaraldehyde, the migrated cells
adhering to the underside of the membrane facing the chemoattractant
were stained with crystal violet. The number of cells that had migrated
through the filter was determined by counting the cells in 5 high-power
fields under a microscope. Three replicate filters were used for each
treatment. As a control, chemotaxis induced by MCP-1 was inhibited by
including a neutralizing mouse anti–human MCP-1 monoclonal
antibody.
Determination of Cellular Cholesterol
Cellular lipids were extracted from 106
cells with chloroform/methanol (2:1, vol/vol), dried under
N2, and resuspended in 50 µL isopropanol. Free
and total cholesterol contents after hydrolysis of
cholesterol ester with cholesterol hydrolase
were estimated by the fluorometric method, and the content of
cholesterol ester was calculated by subtracting free
cholesterol from total
cholesterol.41
Other Analytical Procedures
Protein was determined by the method of Lowry et
al.42 Data are expressed as mean±SD and were
analyzed by Student's t
test.43 Plasma triglycerides and
total, HDL, and LDL cholesterol were analyzed in
the Lipid Analytical Laboratory of the La Jolla Lipid Clinic. The
analytical procedures used have been standardized by the Centers for
Disease Control and Prevention, Atlanta, Ga. Total
cholesterol, HDL cholesterol after removal of
other lipoproteins by heparin/manganese
precipitation,44 and triglycerides
were measured by enzymatic techniques with an Abbott VP
analyzer (Abbott Laboratories) using cholesterol
reagent (Boehringer Mannheim) and triglyceride
reagents (Abbott Laboratories).
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Results |
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Induction of CCR2 Expression by LDL
THP-1 monocytes were routinely grown in RPMI 1640 medium supplemented with 10% serum. The final concentration of total cholesterol in the growth medium was, on average, 80 µg/mL and that of LDL cholesterol was 30 µg/mL, which equates to
15 µg of LDL protein per mL. THP-1 monocytes maintained
under these conditions showed a specific binding of 5.9±0.6 fmol MCP-1
per 106 cells. Treatment with freshly isolated
human plasma LDL at 5 and 50 µg LDL protein per mL for 24 hours
induced a 1.2-fold (P<0.05) and a 1.7-fold, respectively,
increase in CCR2 expression (Figure 1A).
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To determine whether plasma LDL affects gene transcription or
translation, THP-1 monocytes were treated with LDL (5 and 50 µg
protein per mL) for 24 hours, and total RNA was isolated from the
untreated as well as LDL-treated cells. The steady-state level of CCR2
transcripts was estimated by semiquantitative PCR analysis of
reverse-transcribed mRNA as described under Methods. As an internal
control, the housekeeping gene GAPDH was estimated under identical
conditions and used to normalize relative changes in CCR2 mRNA levels.
The presence of human LDL in the culture medium had a profound effect
on CCR2 expression and induced a 2.5- to 3-fold increase of CCR2
transcripts in THP-1 cells (Figure 1B
).
To demonstrate that LDL-mediated CCR2 expression was not peculiar to
THP-1 cells, we determined CCR2 mRNA levels in freshly isolated human
blood monocytes. Four normocholesterolemic females (LDL
cholesterol <100 mg/dL) and 11
hypercholesterolemic females (LDL
cholesterol >130 mg/dL) matched for age were studied. In
all cases, the monocyte CCR2 expression, estimated by semiquantitative
PCR amplification of reverse-transcribed mRNA, was higher in
individuals of the hypercholesterolemic group than in
the ones of the control group (Figure 1C). The plasma lipid profile and
monocyte CCR2 expression of all subjects studied are summarized in
Table 1. On average, the CCR2 expression
in the hypercholesterolemic group was 2-fold higher
compared with the control group. Analysis of the data suggested
a significant correlation between the LDL cholesterol level
and CCR2 mRNA expression.
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The time course of the effect on CCR2 expression was examined by
incubating THP-1 monocytes with LDL (50 µg of protein per mL) for as
long as 72 hours. Surface expression was determined by
125I–MCP-1 binding analysis at the
various time points. By 6 hours, the binding sites for MCP-1 on the
plasma membrane were increased by 20% (P<0.006) and by 48
hours, a doubling of MCP-1 binding was observed (Figure 2). The results from CCR2 mRNA expression
and these binding studies suggested that the LDL-mediated increase in
CCR2 expression was regulated primarily at the level of gene
transcription.
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Receptor surface expression was determined by end-point ligand binding
analysis at saturating ligand concentrations and by assuming an
unchanged binding affinity of 0.5 nmol/L. To determine whether LDL
induced any changes in binding affinity, we generated complete binding
curves with untreated THP-1 cells and with THP-1 cells that had been
treated with LDL (50 µg of protein per mL) for 24 and 48 hours. As
shown in Table 2, the binding affinities
remained essentially unchanged. Consistent with the data
reported above, the number of binding sites for MCP-1 increased
1.7-fold after 24 hours and 2.1-fold after 48 hours. These studies
demonstrated that LDL induces a time-dependent increase in monocyte
CCR2 expression without affecting the binding affinity.
Effect of Increased CCR2 Expression on Chemotaxis
The functional relevance of the increased density of CCR2 on the
cell surface was analyzed by chemotaxis assays. THP-1 monocytes
were incubated for 24 hours in culture medium supplemented with
increasing concentrations of freshly isolated LDL, and the cells'
chemotactic response to MCP-1 and
N-formyl-methionyl-leucyl-phenylalanine was examined. As in
previous experiments, human LDL added at concentrations as low as 5
µg protein per mL induced a 20% increase in CCR2 surface expression
(Figure 1). However, this rather small increase in receptor number had
a profound effect on chemotaxis, almost doubling the response to 10
nmol/L MCP-1 (Figure 3). A close
correlation between LDL concentration in the medium, the number of cell
surface receptors for MCP-1, and the chemotactic responsiveness was
observed. When the basal concentration of LDL in the medium (15 µg
LDL protein per mL, which equates to 30 µg LDL
cholesterol per mL) was increased by 500 µg of LDL
protein per mL (1 mg LDL cholesterol per mL), the
chemotactic response of the treated THP-1 monocytes to 10 nmol/L MCP-1
increased 3-fold compared with the untreated controls. This response
was almost completely abolished by a neutralizing mouse anti–human
MCP-1 antibody. The chemotaxis induced by MCP-1 in cells treated with
500 µg LDL protein per mL was identical to that induced by 10 nmol/L
N-formyl-methionyl-leucyl-phenylalanine. This peptide, a
synthetic analogue of naturally occurring products derived from
bacteria during infection, is commonly used to study activation of
neutrophils and monocytes through the receptor for N-formyl
peptides.45 The chemotactic activity in response
to the N-formyl peptide was not affected by LDL.
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Potential Mechanisms of Lipoprotein-Induced CCR2
expression
To determine whether the effect on CCR2 expression was specific
for native LDL or whether modified forms of LDL had similar effects, we
incubated THP-1 monocytes with mildly and fully oxidized LDL (50 µg
of protein per mL) for as long as 48 hours. The extent of oxidation of
mildly oxidized LDL, prepared by brief incubation with copper, was
similar to that of mildly oxidized LDL described by
others46 and contained 4 nmol TBARS per mg
protein. Fully oxidized LDL prepared by extensive oxidation with copper
contained 55 nmol TBARS per mg protein. No cytotoxicity was observed
at the concentrations of oxidized LDL used, and the proliferation rate
and viability of the THP-1 monocytes remained unchanged. Fully oxidized
LDL rapidly downregulated CCR2 expression, and by 6 hours, the binding
sites for MCP-1 were reduced by 60% (Figure 4A). After 24 hours, essentially no cell
surface expression of CCR2 was detected, and only residual binding of
MCP-1 was observed. Mildly oxidized LDL had no effect on monocyte CCR2
expression, and the binding of 125I–MCP-1
remained unchanged (Figure 4B).
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To test whether the response to LDL required interaction with the LDL
receptor, THP-1 monocytes were incubated for as long as 48 hours in
complete medium supplemented with human LDL (50 µg protein per mL) in
the absence or presence of anti–LDL receptor IgG C7 (1:200 dilution),
an antibody that effectively blocks the interaction of LDL with its
receptor.47 This antibody completely prevented
the LDL-induced expression of CCR2 (Figure 5). To further test whether the effect
was mediated by the LDL receptor, we incubated THP-1 monocytes with
reductively methylated LDL (50 µg of protein per mL) for as long as
48 hours. Reductive methylation of 30% or more of the lysine residues
prevents the binding of LDL to surface
receptors.36 About 80% of the lysine residues
were methylated in our preparation. Although so modified LDL was not
taken up by the native LDL receptor, it induced a small increase in
expression of CCR2 (Figure 5). Note, however, that the increase with
time was not significant compared with the results obtained with native
LDL. Native LDL was avidly taken up by THP-1 monocytes and
significantly increased intracellular cholesterol by 2-
to 3-fold (Figure 6A). There was a strong
correlation between the cellular cholesterol content and
the level of CCR2 expression (cf Figures 5 and 6A). This increase of
LDL-derived intracellular cholesterol was completely
blocked by the presence of the anti–LDL receptor IgG C7 (Figure 6B),
which also prevented any LDL-induced CCR2 expression (Figure 5).
Although modification of lysine residues retards the clearance rate of
LDL in animals, it is eventually removed from the plasma through
unknown mechanisms, perhaps receptor-independent
pinocytosis.34 Methylated LDL may be taken up by
THP-1 monocytes by a similar mechanism, and this possibility might
account for the slightly increased intracellular
cholesterol content (Figure 6C). The magnitude of this rise
paralleled the increase in CCR2 expression (Figure 5), suggesting
that raising cellular cholesterol levels might trigger CCR2
gene expression.
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To test this hypothesis, we incubated THP-1 monocytes with 25 µg/mL
of free cholesterol for 24 hours. This treatment increased
the cellular pool of cholesterol 4-fold (Figure 7A) and caused a 2-fold increase in CCR2
expression (Figure 7B), which was not affected by the anti–LDL
receptor IgG C7 (data not shown). Interestingly, CCR2 surface
expression remained elevated, even after the
cholesterol-loaded cells were returned to normal growth
medium without supplemental cholesterol and after the
cellular cholesterol had dropped significantly.
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Most studies on monocyte recruitment have focused on lipoprotein-induced changes in cells of the arterial wall, whereas the effects of lipoproteins on gene expression in the monocyte have received little attention. Gerrity et al25 showed that a chemotactic factor isolated from atherosclerotic lesions strongly stimulated chemotaxis in monocytes isolated from hypercholesterolemic swine but only weakly in monocytes from control swine. The reported molecular mass of the chemotactic factor was
6000 Da, similar to that of human MCP-1, now known to be
synthesized and secreted by cells in atherosclerotic
lesions.17 18 Similar studies in human subjects
indicated that monocytes isolated from
hypercholesterolemic patients are functionally
different and adhere in larger numbers to cultured
endothelial cells than do monocytes from matched
controls.26
Our findings suggest that the increased monocyte accumulation in the
vessel wall during atherogenesis may result in part from an enhanced
chemotactic response due to upregulated CCR2 gene expression. THP-1
monocytes express, on average, 5000 MCP-1 receptors per cell, which is
comparable to the number established for freshly isolated human
monocytes.48 49 A finely tuned network of
cytokines regulates the expression of CCR2. The transformation
of monocytes from an MCP-1–responsive state to a state in which
monocytes no longer respond to MCP-1 is caused by a reduction of CCR2
expression triggered by certain proinflammatory
cytokines.23 In contrast, basal CCR2
surface expression was increased by plasma LDL in a time- and
concentration-dependent manner. Analysis of CCR2 message
indicated that the primary factors for the increased CCR2 expression
include augmented rate of gene transcription and changes in processing
or stability of the mRNA. Translational regulation, receptor
sequestration, and recycling from intracellular stores
represent other possible mechanisms by which receptor surface
density can be varied. By 6 hours of treatment with LDL, the number of
CCR2 molecules on the cell surface increased by 20%. De novo
protein synthesis characteristically requires more time, and these
receptors most likely represent a population that was recruited
from intracellular stores. However, the major increase of CCR2 protein,
measured by MCP-1 binding, was observed during the subsequent 24 to 48
hours, which is characteristic for de novo protein synthesis.
Chemotaxis is one of the main responses of monocytes triggered by MCP-1.48 The initial binding of MCP-1 is followed by transmembrane signaling, amplification of the primary stimulus, and subsequent induction of cellular responses. CCR2 expression in monocytes is low compared with the 50 000 to 100 000 receptors for N-formyl-methionyl-leucyl-phenylalanine. Although the receptor occupancy necessary for optimal chemotactic response to MCP-1 is unknown, if one considers the relatively low basal expression of CCR2 (5000 receptors per cell), small changes in receptor number can profoundly affect chemotactic activity. In a recent study, we have demonstrated negative control of CCR2 gene expression by proinflammatory cytokines,23 which is associated with a decrease in chemotactic activity. In contrast, a 20% increase of CCR2 on the cell surface, induced by the lowest LDL concentration tested, almost doubled the chemotactic response of THP-1 monocytes to MCP-1. A close correlation between LDL concentration in the medium, the number of receptors for MCP-1 on the cell surface, and the chemotactic response was observed. Up to 3-fold increases in monocyte chemotactic activities were recorded at increasing LDL concentrations. Interestingly, the chemotactic response of the cells to N-formyl-methionyl-leucyl-phenylalanine remained unchanged by the LDL treatment. The receptor for this chemotactic peptide is either not affected by LDL or the basal receptor expression, which is at least 10-fold higher than that of CCR2, is already sufficient for optimal chemotaxis.
Leukocyte extravasation is a multistep process that involves reversible rolling, firm adhesion, and transendothelial migration. Several of the 7 transmembrane-spanning chemoattractant receptors have been shown to support integrin-mediated firm adhesion of leukocytes to the endothelium.50 Thus, an increase in CCR2 expression as seen after exposure to high plasma LDL levels may support sustained, firm adhesion of monocytes to the vessel wall and cause excessive monocyte recruitment. This hypothesis was confirmed by results from a recent study on CCR2-deficient mice, demonstrating that CCR2 is not only essential for monocyte chemotaxis but also might play an important role in the firm adhesion of monocytes to the endothelium preceding diapedesis.51
Recent studies have demonstrated that LDL is capable of eliciting intracellular signaling responses in a variety of cells.52 53 However, the effect of LDL on CCR2 expression appears independent of such signaling events and is primarily caused by the cellular levels of cholesterol. The exact mechanism by which cholesterol controls the biosynthesis of CCR2 is still unknown and will be the topic of future studies. Analysis of exon 1 of CCR222 revealed the presence of a sterol-regulatory element–like sequence, CAACGCAC, in the 5' untranslated region, suggesting that sterols may play an important role in CCR2 gene regulation.
Oxidized LDL, found mainly in atherosclerotic lesions, is rapidly taken up by monocytes/macrophages through scavenger receptor–mediated endocytosis.54 55 56 57 58 Although this process causes massive accumulation of cellular cholesterol, it had the opposite effect on CCR2 expression. In contrast to LDL, oxidized LDL induced a rapid loss of monocyte CCR2. Oxidized LDL has been shown to stimulate monocyte-macrophage differentiation,59 probably by inducing cytokine secretion. From a previous study, we know that cytokine-induced activation or differentiation of monocytes resulted in a reduction of CCR2 expression,23 which may explain the effect of oxidized LDL. The extent to which LDL was oxidized largely determined its effect on CCR2 expression, and mildly oxidized LDL did not influence CCR2 expression. The effect of cholesterol on CCR2 expression may be blunted by some other components of mildly oxidized LDL, and no net change in CCR2 expression was observed.
In summary, we have demonstrated that levels of LDL that are characteristic for hypercholesterolemia cause increased CCR2 expression and chemotactic activity in monocytes. Cytokines modulate the immune response by regulating the secretion of chemokines as well as by controlling CCR2 expression.23 High LDL concentrations in the plasma may disturb the delicate regulation of CCR2 expression by these cytokines and may result in excessive accumulation of monocytes in the arterial wall.
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Acknowledgments |
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This work was supported in part by National Institutes of Health (Bethesda, Md) grant HL56989 (SCOR in Molecular Medicine and Atherosclerosis) and by grant 6IT-0133 from the Tobacco-Related Disease Research Program, California (to O.Q.). Ki Hoon Han was supported by a fellowship from the Shim-Jae Culture Foundation, Korea. We thank Drs D. Steinberg and J.L. Witztum for helpful discussions and F. Almazan and N. Kondratenko for excellent technical assistance. We are grateful to Dr Ki Ok Han for providing us with human samples.
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Footnotes |
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K.H.H. and R.K.T. contributed equally to this work.
Received April 7, 1998; accepted August 3, 1998.
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References |
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R. J. Aiello, D. Brees, P.-A. Bourassa, L. Royer, S. Lindsey, T. Coskran, M. Haghpassand, and O. L. Francone Increased Atherosclerosis in Hyperlipidemic Mice With Inactivation of ABCA1 in Macrophages Arterioscler. Thromb. Vasc. Biol., April 1, 2002; 22(4): 630 - 637. [Abstract] [Full Text] [PDF]
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