Download Oxidized LDL-Containing Immune Complexes Induce Fc Gamma

Oxidized LDL-Containing Immune Complexes Induce Fc
Gamma Receptor I–Mediated Mitogen-Activated Protein
Kinase Activation in THP-1 Macrophages
Yan Huang, Ayad Jaffa, Sinikka Koskinen, Akira Takei, Maria F. Lopes-Virella
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Abstract—Our previous studies have shown that Fc gamma receptor (FcgR)-mediated uptake of LDL-containing immune
complexes (oxLDL-ICs) by human monocyte-derived macrophages leads to not only transformation of macrophages
into foam cells but also macrophage activation and release of cytokines. It has been shown that cross-linking of FcgR
triggers activation of signal transduction pathways that alter gene expression in macrophages. In this study, we
determined whether engagement of FcgR by oxLDL-ICs leads to activation of mitogen-activated protein (MAP) kinase
pathway, a signaling cascade serving many important functions, including the regulation of gene expression, in THP-1
macrophage-like cells. Our results from immunoblotting, using specific anti-phosphorylated MAP kinase antibodies,
showed that oxLDL-ICs induced extracellular signal regulated kinase 2 (ERK2) MAP kinase phosphorylation in THP-1
macrophage-like cells in time- and dose-dependent manners. Cholesterol loading before stimulation led to a longer
phosphorylation of ERK2. Nuclear translocation of phosphorylated ERK was markedly increased after the stimulation.
Moreover, our data showed that oxLDL-IC induction of MAP kinase was prevented by human monomeric IgG1,
suggesting that the specific engagement of type I FcgR by oxLDL-IC is responsible for the MAP kinase activation.
Finally, we showed that human anti-oxLDL autoantibody-containing immune complexes immobilized on type I collagen
induced MAP kinase activation in THP-1 cells. These results strongly suggest that oxLDL-IC, which has been detected
in atherosclerotic plaques, may play an important role in macrophage activation and atherogenesis. (Arterioscler
Thromb Vasc Biol. 1999;19:1600-1607.)
Key Words: oxidized LDL n immune complex n mitogen-activated protein kinase n Fc gamma receptor
TNFa and IL-1b have a considerable impact in the development of atherosclerosis.12–14
In our previous studies investigating the mechanisms by
which LDL-ICs induced macrophage activation and foam cell
formation, we demonstrated that both processes depended on
engagement of FcgRI by LDL-IC.8 It has been documented
that cross-linking of FcgRI triggers signal transduction pathways that modulate macrophage functions.15–17 Among these
pathways, those that activate kinases belonging to the
mitogen-activated protein (MAP) kinase family have received particular attention, because many of these enzymes
translocate to the cell nucleus to regulate gene expression.18
Recent studies have shown that MAP kinase pathway mediates extracellular stimulus-altered macrophage functions such
as lipopolysaccharide-stimulated TNF-a expression.19 –21
These observations prompted us to investigate whether engagement of FcgRI by oxLDL-ICs induces activation of
MAP kinase in macrophages.
Our results showed that oxLDL-ICs induced MAP kinase
phosphorylation in THP-1 macrophage-like cells in time- and
concentration-dependent manners and that prior cholesterol-
I
n recent years, circulating autoantibodies against modified
forms of LDL and immune complexes containing modified
LDL have been detected in diabetic and nondiabetic subjects,
with and without coronary artery disease, by our and other
research groups.1–5 Several lines of evidence suggest that
autoantibodies against oxidized LDL (oxLDL) and the immune complexes formed by the association of these autoantibodies with oxLDL (oxLDL-ICs) play an important role in
the development of atherosclerosis: (1) a correlation between
the titer of autoantibodies to oxLDL and the rate of progression of atherosclerosis has been shown to be statistically
significant3; (2) both anti-oxLDL autoantibodies and oxLDLICs have been detected in human atherosclerotic plaques6;
and (3) it has been shown that uptake of immune complexes
containing either native or oxidized LDL by macrophages
through type I Fc gamma receptor (FcgRI) led to the
transformation of macrophages into foam cells7–10 and to their
activation.11 Activation of macrophages by oxLDL-IC, in
turn, results in increased cytokine release of, for example,
tumor necrosis factor a (TNFa) and interleukin-1b (IL-1b),
and enhanced respiratory burst.11 It has been shown that
Received August 3, 1998; revision accepted December 11, 1998.
From the Division of Endocrinology, Diabetes and Medical Genetics, Department of Medicine, Medical University of South Carolina (Y.H., A.J., S.K.,
A.T., M.F.L.-V.); and Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC.
Correspondence to Maria F. Lopes-Virella, MD, PhD, Division of Endocrinology, Diabetes and Medical Genetics, Department of Medicine, Medical
University of South Carolina, 114 Doughty St, Charleston, SC 29403. E-mail [email protected]
© 1999 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
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Huang et al
Oxidized LDL Immune Complexes Stimulate MAP Kinase
loading of the cells induced a more sustained MAP kinase
activation. Engagement of FcgRI by oxLDL-ICs was required for the activation of MAP kinase because blocking of
FcgRI with monomeric IgG1 precluded MAP kinase activation. Finally, we showed that after immobilization by binding
to type I collagen, immune complexes containing purified
human anti-oxLDL autoantibodies, like insoluble immune
complexes prepared with rabbit anti-oxLDL antibodies, induced FcgRI-mediated MAP kinase activation in THP-1
macrophage-like cells.
Methods
Materials
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Rabbit anti-phosphorylated and anti-p42/p44 MAP kinase antibodies
were purchased from New England Biolabs, Inc. Antiphosphotyrosine antibody was purchased from Transduction Laboratories. CNBr-Sepharose 4B columns were purchased from Pharmacia Biotech. Polyvinylidene difluoride (PVDF) membranes and
chemiluminescence reagents were purchased from NEN Life
Science Products.
Cell Culture
The human monocytic cell line THP-1 was obtained from the
American Type Culture Collection. The cells were cultured in a 5%
CO2 atmosphere in Iscove’s modified Dulbecco’s medium (IMDM)
supplemented with 10% FCS. The THP-1 cells were transformed
into macrophage-like cells by treatment with 0.16 mmol/L phorbol
12-myristate 13-acetate (PMA) at 37°C for 24 hours.22 After PMA
treatment, THP-1 cells were washed 3 times with PBS and incubated
with fresh IMDM containing 10% FCS for 24 hours. The cells were
then stimulated with oxLDL-ICs in serum-free medium (SFM).
Lipoprotein Isolation and Modification
Blood for lipoprotein isolation was collected from normal healthy
volunteers in 0.4 mmol/L EDTA after 12 hours of fasting. Plasma
from 3 to 4 normal volunteers was used for separation of LDL by
preparative ultracentrifugation at 50 000 rpm for 17 hours on a
Beckman L-80 ultracentrifuge after density adjustment with potassium bromide (1.019,density,1.063 g/mL) using a type 70 titanium rotor.9 Isolated LDL was washed by ultracentrifugation,
dialyzed against a 0.15 mol/L sodium chloride solution containing
300 mmol/L EDTA, pH 8.0, passed through an Acrodisc filter
(0.22-mm pore size) to sterilize and remove aggregates, and stored
under nitrogen in the dark at 4°C.
Oxidation of LDL was performed according to the protocol
described by Lopes-Virella et al.8 Briefly, EDTA was removed by
passing LDL over a PD-10 column (Pharmacia Biotech) and after
EDTA removal, Cu21 was added at a final concentration of
10 mmol/L to the LDL preparation and the mixture incubated at 37°C
for 18 hours. To stop the oxidation reaction 200 mmol/L EDTA and
40 mmol/L butylated hydroxyl toluene (BHT) were added, and the
degree of LDL oxidation was determined by measuring the amount
of conjugated dienes formed. Acetylated LDL was prepared as
previously described.23
Preparation of Insoluble Immune Complexes
OxLDL-IC and native LDL-IC (nLDL-IC) were prepared as described previously.9 Briefly, insoluble oxLDL-IC and nLDL-IC were
prepared by incubating overnight, at 4°C, oxLDL (150 mg/mL) or
native LDL (170 mg/mL) with a rabbit anti-LDL antiserum (500
mg/mL) raised in our laboratory. The concentrations of oxLDL or
native LDL and anti-LDL antibody used to prepare the immune
complexes were determined by a precipitin curve, constructed by
incubating 500-mg aliquots of the antiserum with varying amounts of
oxLDL or native LDL and determining the amount of oxLDL or
native LDL that induced the highest degree of immune complex
formation as determined by nephelometry.8 The antigen to antibody
ratio yielding the highest amount of precipitate was used for the
preparation of the insoluble oxLDL-IC and nLDL-IC. The insoluble
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oxLDL-IC or nLDL-IC were washed 3 times and resuspended with
PBS. The protein content of oxLDL-IC and nLDL-IC was determined by Lowry assay.24
Measurement of Free and Esterified
Cholesterol Content
Lipids in THP-1 macrophages were extracted with isopropanol/
hexane (2:3, vol/vol) as previously described.8 Free and total
cholesterol were assayed on a gas chromatograph equipped with an
H2 flame ionization detector. A fused silica capillary column packed
with DB17 was used for the chromatographic separation. Cholesteryl
ester (CE) levels were calculated by subtracting free cholesterol from
total cholesterol levels. b-Stigmasterol was used as an internal
standard.
Preparation of Human IgG1
Human monomeric IgG1 was isolated from the serum of a patient
with an IgG1 multiple myeloma, using a technique previously
described.8 After protein precipitation with ammonium sulfate, the
resuspended protein was subjected to affinity chromatography on a
protein A/G column (ImmunoPure), following the instructions from
the manufacturers.8 After purification, IgG1 was stored at 270°C.
Before the performance of the FcgRI blocking experiments, IgG1
was centrifuged at 100 000g for 30 minutes and immediately added
to the culture medium to ensure complete elimination of aggregates.
Monomeric IgG1 binds to FcgRI with high affinity and does not bind
to any of the other FcgR subtypes.
Subcellular Fractionation
Subcellular fractionation was performed as previously described.25
Briefly, THP-1 macrophage-like cells were washed with PBS and
harvested by scraping with a rubber policeman. The cells were
centrifuged at 1000g at 4°C for 10 minutes and homogenized (10
strokes) with a Dounce homogenator in a buffer containing
10 mmol/L HEPES, pH 7.9, 1.5 mmol/L MgCl2, 10 mmol/L KCl,
0.5 mmol/L DTT, and 2 mmol/L sodium vanadate. The homogenates
were centrifuged at 4000g for 10 minutes, and the supernatants
containing cytosolic protein were collected. The pellets were resuspended and homogenized at 4°C in a buffer containing 20 mmol/L
HEPES, pH 7.9, 25% glycerol, 420 mmol/L NaCl, 0.2 mmol/L
EDTA, 0.5 mmol/L DTT, 0.5 mmol/L PMSF, and 2 mmol/L sodium
vanadate. The homogenates were then centrifuged at 4°C, 16 000g
for 30 minutes, and the supernatants containing the nuclear proteins
were collected.
Western Blot Analysis
Western blot analysis of MAP kinase was performed with a
PhosphoPlus MAPK Antibody kit, following the instruction from the
manufacturer (New England Biolabs). Briefly, THP-1 macrophagelike cells after oxLDL-IC stimulation were lysed with a lysis buffer
containing 20 mmol/L Tris, pH 8.0, 130 mmol/L NaCl, 10%
glycerol, 10 mmol/L CHAPS, 100 mU/mL aprotinin, 0.156 mg/mL
benzamidine, 2 mmol/L sodium vanadate, and 1 mmol/L PMSF. The
cell protein lysate was electrophoresed in 10% polyacrylamide gel
and transferred to a PVDF membrane (NEN Life Science Products).
Afterward, the membrane was incubated for 1 hour at room temperature with a blocking buffer containing 20 mmol/L Tris, pH 7.6,
130 mmol/L NaCl, 0.1% Tween-20, and 5% nonfat dry milk. The
membrane was then incubated, at 4°C overnight, with primary
antibody (anti-phosphorylated or anti-p42/p44 MAP kinase antibodies), followed by incubation, for 1 hour at room temperature, with
horseradish peroxidase-conjugated sheep anti-rabbit IgG. MAP kinase was visualized by incubating the membrane with chemiluminescence reagents (NEN Life Science Products) for 1 minute and
then exposing it to x-ray film for 30 seconds. The films were scanned
by a densitometer to quantify MAP kinase.
Isolation of Human Anti-OxLDL Antibodies
Antibodies to oxLDL were isolated from human sera by affinity
chromatography as described previously.26 Briefly, LDL was immobilized to CNBr-Sepharose 4B (Pharmacia Biotech) and oxidized by
incubation at 37°C for 18 hours with 10 mmol/L CuCl2. To isolate
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Arterioscler Thromb Vasc Biol.
July 1999
oxLDL antibodies, 1 mL of serum diluted with 4 mL of bicarbonate
buffer was loaded onto the column, and the serum-loaded column
was incubated overnight at 4°C. Unbound proteins were washed out
with the equilibrating buffer, and 2 bound fractions were eluted in
sequence, the first with 0.1 mol/L NaHCO3 buffer containing 0.5
mol/L NaCl, pH 8.3, and the second with 0.5 mol/L acetate buffer
also containing 0.5 mol/L NaCl, pH 4.0.
Stimulation of MAP Kinase by Immune
Complexes Containing Human
Anti-OxLDL Antibodies
OxLDL (100 mg/mL) was incubated at 37°C for 24 hours in 35-mm
tissue culture dishes precoated with type I collagen (1 mg/mL,
Sigma). After removing the unbound oxLDL by washing 3 times
with PBS, human anti-oxLDL autoantibodies (200 mg/mL) were
added to each well, and the wells were incubated for another 24
hours at 4°C. The dishes were then washed 3 times with PBS. THP-1
cells suspended in SFM (2.73106/dish) were seeded into each well.
The cells were lysed 10, 20, 30, 40, or 60 minutes after seeding, and
the cell lysates were used for Western blot analysis of MAP kinase
as described above.
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Results
Time-Dependent Stimulation of MAP Kinase in
THP-1 Macrophage-Like Cells by OxLDL-ICs
THP-1 macrophage-like cells were incubated with 150
mg/mL of oxLDL-IC for 0, 10, 20, 30, 40, or 60 minutes, and
afterward the cells were lysed as described in the Methods.
The cell lysates were then subjected to Western blot analysis
of phosphorylated and total MAP kinase with anti-phosphorylated and anti-p42/p44 MAP kinase antibodies, respectively.
Results showed that oxLDL-IC significantly induced extracellular signal regulated kinase 2 (ERK2) (p42) phosphorylation in THP-1 macrophage-like cells in a time-dependent
manner, with peak stimulation at 20 minutes and a quick
return to near baseline levels by 40 minutes (Figure 1). As a
control, the amount of total MAP kinase, including phosphorylated and nonphosphorylated p42/p44 MAP kinase, was
determined, and it did not change after oxLDL-IC
stimulation.
Concentration-Dependent Stimulation of MAP
Kinase by OxLDL-ICs
THP-1 macrophage-like cells were incubated with 0, 25, 50,
100, 150, or 200 mg/mL of oxLDL-IC for 20 minutes, and the
cells were lysed after the incubation. Western blot analysis of
the cell lysates using antibodies to specifically identify
phosphorylated and total MAP kinase was performed. As
shown in Figure 2, oxLDL-IC stimulated p42 MAP kinase
phosphorylation in a concentration-dependent manner. The
phosphorylation reached its peak when the cells were stimulated with 100 mg/mL of oxLDL-IC. Higher levels of
oxLDL-IC had a similar effect. Therefore, we selected 150
mg/mL of oxLDL-IC as the optimal concentration to stimulate cells in the subsequent experiments.
Figure 1. Time-dependent stimulation of MAP kinase in THP-1
macrophage-like cells by oxLDL-ICs. THP-1 cells were incubated with IMDM containing 10% FBS and 160 nmol/L PMA for
24 hours. After washing, the cells were incubated for 24 hours
with culture medium and thereafter stimulated with 150 mg/mL
of oxLDL-ICs for the times indicated and lysed as described in
Methods. Twenty-five micrograms of cell protein was electrophoresed on 10% SDS PAGE and transferred to a PVDF membrane. The membrane was immunoblotted with anti-phosphorylated and anti-p44/p42 MAP kinase antibodies to determine
phosphorylated and total MAP kinase as described in Methods.
MAP kinase was visualized by incubation of the PVDF membrane with chemiluminescence reagent for 1 minute and exposure to x-ray film for 15 seconds. The cellular MAP kinase level
was quantified by densitometry scanning of the film. Data are
representative of 3 experiments with similar results.
cells with acetyl-LDL for 48 hours. As shown in Figure 3,
cellular CE content increased 26-fold in acetyl-LDL-treated
cells as compared with that in untreated cells. After CE
loading, the cells were incubated with either medium alone or
medium containing 150 mg/mL of oxLDL-IC, and MAP
kinase phosphorylation was determined. As shown in Figure
4, oxLDL-ICs induced p42 MAP kinase phosphorylation in
CE-laden cells in a time-dependent manner. Maximal phosphorylation was observed at 30 minutes, and the phosphorylation did not return to the baseline level at 120 minutes.
Thus, phosphorylation of p42 MAP kinase in response to
oxLDL-IC stimulation was slightly delayed, but remained
increased over the baseline level for a substantially longer
period in cholesterol-loaded cells when compared with cells
not previously loaded with cholesterol.
Stimulation of MAP Kinase in CE-Laden THP-1
Macrophage-Like Cells by OxLDL-ICs
Because macrophage foam cells are the predominant cells in
atherosclerotic plaques, it is important to determine whether
oxLDL-ICs stimulate MAP kinase in CE-laden THP-1 macrophage-like cells. To load the cells with cholesterol, we
treated THP-1 cells with PMA for 3 days to maximize
scavenger receptor expression23 and afterward incubated the
Figure 2. Dose-dependent stimulation of MAP kinase in THP-1
macrophage-like cells by oxLDL-IC. THP-1 macrophage-like
cells were stimulated for 20 minutes with different concentrations of oxLDL-ICs as indicated and then lysed. Phosphorylation
of MAP kinase in the cells was analyzed by immunoblotting as
described in Methods. The above data are representative of 3
experiments with similar results.
Huang et al
Oxidized LDL Immune Complexes Stimulate MAP Kinase
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Figure 3. Accumulation of CE in THP-1 macrophage-like cells
incubated with acetyl-LDL (acLDL). THP-1 cells were preincubated with PMA (160 nmol/L) for 3 days, followed by incubation
with 100 mg/mL of acetylated LDL for 2 days. Cellular lipids
were then extracted as described in Methods. Total and free
cholesterol contents were determined by gas chromatography
as described in Methods. CE content was calculated by subtracting free from total cholesterol. Data represent the
mean6SD of 3 experiments run in triplicate.
Stimulation of Tyrosine Phosphorylation of
Cellular Proteins in THP-1 Macrophage-Like Cells
by OxLDL-ICs
MAP kinase cascade activation triggered by oxLDL-ICs
leads to phosphorylation of p42 MAP kinase. Thus, tyrosine
phosphorylation of other proteins, such as Shc or Raf-1, in the
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Figure 5. Time course of tyrosine phosphorylation in CE-laden
THP-1 macrophage-like cells stimulated with oxLDL-ICs. The
CE-laden THP-1 macrophage-like cells were incubated with
SFM or SFM containing 150 mg/mL of oxLDL-ICs for various
periods as indicated in Figure 3. After the incubation, cell
lysates were prepared and submitted to immunoblotting using
the anti-phosphorylated tyrosine antibody RC20 (Transduction
Laboratories) as described in Methods. The membrane was
incubated with chemiluminescence reagent for 1 minute and
exposed to x-ray film for 15 seconds to detect tyrosine phosphorylation. The data presented are representative of 3 experiments with similar results.
upstream MAP kinase cascade is required for the downstream
MAP kinase activation.16 Therefore, we expected that in
addition to the p42 MAP kinase, oxLDL-ICs would also
induce tyrosine phosphorylation of other proteins in the MAP
kinase cascade. Furthermore, OxLDL-ICs may also induce
tyrosine phosphorylation of proteins in other signaling pathways. To study the effects of oxLDL-ICs on cellular tyrosine
phosphorylation, the samples prepared for the MAP kinase
study (Figure 4) were analyzed by immunoblotting using
RC-20, an anti-phosphotyrosine antibody that recognizes
phosphorylated tyrosine residues. Our data (Figure 5) showed
that treatment of CE-laden THP-1 macrophage-like cells with
oxLDL-IC resulted in a marked enhancement of tyrosine
phosphorylation in several cellular proteins. Tyrosine phosphorylation was observed in proteins of 120, 80, 70, and 42 to
44 kDa. Peak stimulation was observed at 30 minutes, similar
to the time for p42 MAP kinase phosphorylation in CE-laden
THP-1 macrophages (Figure 4).
Nuclear Translocation of MAP Kinase in
CE-Laden THP-1 Macrophages Induced
by OxLDL-ICs
Nuclear translocation of MAP kinase, as a consequence of
MAP kinase phosphorylation, was studied by Western blot
analysis in nuclear extracts isolated from CE-laden THP-1
macrophage-like cells incubated with or without oxLDL-ICs.
Results showed a marked increase in both cytoplasm and
nuclear phosphorylated p42 MAP kinase after stimulation
with oxLDL-ICs for 30 minutes (Figure 6). These data
confirmed that oxLDL-ICs induced ERK MAP kinase activation in CE-laden THP-1 macrophage-like cells.
Figure 4. Time course of MAP kinase phosphorylation in
CE-laden THP-1 macrophage-like cells stimulated with oxLDLICs. THP-1 macrophage-like cells were treated with 100 mg/mL
of acetylated LDL to induce CE accumulation as described
above. The cells were then stimulated with 150 mg/mL of
oxLDL-ICs for the periods indicated. Twenty-five micrograms of
cellular proteins was subjected to immunoblotting using antiphosphorylated MAP kinase antibodies to determine phosphorylation of MAP kinase as described in Methods. MAP kinase was
detected by incubation of the PVDF membrane with a chemiluminescence reagent for 1 minute and exposure to x-ray film for
15 seconds. The MAP kinase level was quantified by densitometry scanning of the film. The data presented are representative
of 3 experiments with similar results.
Engagement of Type I FcgR by OxLDL-ICs Is
Required for MAP Kinase Phosphorylation
We have previously shown that LDL-ICs are taken up by
THP-1 cells through FcgRI.8 To determine whether engagement of FcgRI by oxLDL-IC was responsible for MAP
kinase phosphorylation, we performed 2 types of experiments. First, we incubated THP-1 cells with oxLDL-ICs in
the presence of either human monomeric IgG1, which binds
with high affinity to FcgRI, or anti-CD32 monoclonal antibody (IV3), which binds to type II FcgR. The results showed
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Arterioscler Thromb Vasc Biol.
July 1999
Figure 6. Nuclear translocation of MAP kinase in oxLDL-IC–
stimulated THP-1 macrophage-like cells. THP-1 macrophagelike cells were incubated in SFM or SFM supplemented with 150
mg/mL of oxLDL-ICs for 30 minutes, and nuclear and cytosol
proteins were isolated as described in Methods. Twenty-five
micrograms of nuclear and cytosol proteins was subjected to
Western blot analysis of phosphorylated MAP kinase with antiphosphorylated MAP kinase antibodies as described in Methods. Phosphorylated MAP kinase was visualized by incubating
the membrane with chemiluminescence reagent for 1 minute
and exposing it to x-ray film for 15 seconds.
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that human IgG1 blocked MAP kinase phosphorylation in a
dose-dependent manner with 90% inhibition at 10 mg/mL,
whereas anti-CD32 monoclonal antibodies had no effect
(Figure 7). These data suggest that engagement of FcgRI is
required for MAP kinase activation by oxLDL-IC. Second,
we stimulated the cells with nLDL-IC to induce MAP kinase
phosphorylation. Results showed that nLDL-ICs also stimulated MAP kinase in a time-dependent manner similar to that
observed with oxLDL-IC (Figure 8). Inasmuch as our previous study has shown that nLDL-IC engaged FcgRI in THP-1
macrophages,8 these data further confirm that the engagement
of FcgRI is required for MAP kinase activation.
Immune Complexes Containing Human
Autoantibodies to OxLDL Stimulate MAP Kinase
in THP-1 Cells
Human anti-oxLDL autoantibodies have been isolated from
serum and characterized in our laboratories.26 Preparation of
insoluble immune complexes using human antibodies is not
feasible because of their low affinity to oxidized LDL. Based
on the studies showing that the positively charged collagenous domain of the scavenger receptor I and II serves as the
binding site for oxLDL27 and that oxLDL binds to type I
collagen more efficiently than to other types of collagens in
vitro,28,29 we immobilized oxLDL on wells coated with type
I collagen as described in the Methods. After immobilization
of ox-LDL on the collagen, purified human anti-oxLDL
autoantibodies were added to the wells to bind to oxLDL.
After washing out the unbound antibodies, THP-1 cells were
seeded into the wells and incubated for periods ranging from
0 to 50 minutes. The cells were then lysed, and phosphory-
Figure 7. Blocking of oxLDL-IC–stimulated MAP kinase phosphorylation by monomeric human IgG1. THP-1 macrophage-like
cells were incubated with 150 mg/mL of oxLDL-ICs for 30 minutes in the presence of increasing concentrations of monomeric
human IgG1 or of anti-FcgRII (CD32) monoclonal antibodies
(IV3) as indicated. The cells were lysed after the incubation, and
25 mg of cell proteins was used for Western blot analysis of
MAP kinase phosphorylation as described in Methods.
Figure 8. Stimulation of MAP kinase by immune complexes
containing native LDL. THP-1 macrophage-like cells were incubated with 150 mg/mL of native LDL-containing immune complexes for 0, 5, 10, 20, or 30 minutes and then lysed. Twentyfive micrograms of cell proteins was subjected to Western blot
analysis of MAP kinase phosphorylation with anti-phosphorylated MAP kinase as described in Methods. Total MAP kinase was
detected by Western blot analysis with anti-p42/p44 MAP
kinase antibodies.
lation of MAP kinase was assessed by Western blot analysis.
THP-1 cells seeded into wells that lacked either oxLDL or
human anti-oxLDL autoantibodies served as negative controls. Our results showed that the immune complexes immobilized on type I collagen induced phosphorylation of p42
MAP kinase in a time-dependent manner, with maximal
phosphorylation at 20 minutes (Figure 9A) and that the
phosphorylation was also blocked by human monomeric
IgG1 (Figure 9B). In contrast, MAP kinase was not induced
in the wells missing either oxLDL or anti-oxLDL antibody
(data not shown).
Discussion
The role of oxLDL-IC in atherogenesis has been the subject
of intensive investigation by our and other research groups.1–7
Our in vitro studies have shown that stimulation of human
monocyte-derived macrophages with insoluble LDL-ICs led
to not only the transformation of these cells into foam cells,7
but also their activation.11 Activated macrophage foam cells
released cytokines, which have been shown to contribute to
atherosclerosis by stimulating endothelial procoagulant activity, expression of cell adhesion molecules in the endothelium,
Figure 9. Stimulation of MAP kinase by immune complexes
containing human anti-oxLDL autoantibodies. OxLDL (100
mg/mL) was incubated at 37°C for 24 hours in 35-mm tissue
culture dishes precoated with type I collagen (1 mg/mL). After
washing with PBS 3 times, the dishes were incubated with
human anti-oxLDL autoantibodies (200 mg/mL) at 4°C for 24
hours. The dishes were washed with PBS 3 times to remove
antibodies not bound to oxLDL, and THP-1 cells (2.73106/dish)
were seeded into the dishes in SFM. The cells were lysed 10,
20, 30, 40, or 60 minutes after the seeding, and the cell lysate
was subjected to Western blot analysis of MAP kinase (A) as
described in Methods. For the blocking study, THP-1 cells were
preincubated with monomeric human IgG1 for 10 minutes
before seeding the cells into the culture dishes (B).
Huang et al
Oxidized LDL Immune Complexes Stimulate MAP Kinase
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and smooth muscle cell proliferation.12–14,30 We have recently
shown that LDL-ICs preferentially engaged FcgRI in human
macrophages and that this engagement of FcgRI is required
for activation of these cells.8 Thus, it is likely that LDL-IC–
induced macrophage activation is mediated by FcgRI-linked
signal transduction pathways. Of these signaling pathways,
the ERK MAP kinase, a member of the MAP kinase family,
plays an important role in the modulation of multiple cellular
functions during macrophage activation.18 Recent studies
have shown that the ERK MAP kinase pathway mediates
lipopolysaccharide-stimulated biosynthesis and secretion of
TNF20 and TNFa-stimulated insulin-like growth factor-1
synthesis31 in activated macrophages. Thus, it is important to
examine the effect of oxLDL-IC on ERK MAP kinase
activation in macrophages.
The present study demonstrated that oxLDL-IC–induced
stimulation of MAP kinase, mainly p42 or ERK2, in THP-1
macrophage-like cells is time- and concentration-dependent.
Of great interest are our findings showing that oxLDL-ICs
stimulated MAP kinase phosphorylation in CE-laden THP-1
macrophages. Recent evidence indicated that macrophage
foam cells play a major role in the initiation and progression
of atherosclerotic lesions.10,32 In response to cholesterol
loading, foam cells alter their metabolic functions, such as
increasing expression of phospholipids,33 apoE,34 tissue factor,35 matrix metalloproteinases,36 and monocyte chemoattractant protein 1.37 Although the mechanism by which gene
expression is altered in foam cells is not well defined, it has
been postulated that cell lipid content can regulate cellular
signaling and, as a consequence, alter gene expression in
response to extracellular stimuli.38 Our present study has
shown that the phosphorylation of MAP kinase in CE-laden
THP-1 macrophage-like cells remained significantly longer
(90 minutes) than that in cells without cholesterol loading (10
minutes), suggesting that the cellular cholesterol content
could affect the process of MAP kinase activation in macrophages. It has been reported recently that the foam cells
induced by treatment of THP-1 macrophages with oxLDL
have lipid-swollen lysosomes and Golgi hypertrophy that are
similar to that observed in atherosclerotic foam cells,39
suggesting that the foam cells induced by oxLDL in vitro are
a relevant model for atherosclerotic foam cells. In the present
study, however, considering that oxLDL also stimulates MAP
kinase in macrophages20 and that cholesterol loading of the
cells with oxLDL will lead to a prestimulation of MAP
kinase, we used acetylated LDL for cholesterol loading.
Another finding in the present study is that engagement of
FcgRI by oxLDL-IC is required to induce oxLDL-IC–
mediated MAP kinase activation. It has been shown that
cross-linking of FcgRI in macrophages by immune complexes elicited signal transduction pathways, including MAP
kinase cascade.16 However, oxLDL has been also shown to
induce MAP kinase activation in macrophages.20 Thus, MAP
kinase can be activated in macrophages by either FcgRIdependent or -independent mechanisms. The mechanism
involved in the activation of MAP kinase when macrophages
were stimulated with antibody-complexed oxLDL was determined by using human monomeric IgG1, which specifically
binds to FcgRI with high affinity, and an anti-CD32 monoclonal antibody (IV3),8 to block the binding of FcgRI and
FcgRII, respectively, by oxLDL-IC. These monomeric anti-
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bodies are unable to cross-link FcgRs and therefore, they do
not elicit MAP kinase activation. Our data showed that
human IgG1 blocked oxLDL-IC stimulated MAP kinase
activation in a concentration-dependent fashion whereas antiCD32 had no effect, suggesting that the activation of MAP
kinase by oxLDL-IC is FcgRI-dependent. These results are
consistent with our previous finding8 that FcgRI was predominantly involved in the binding and uptake of nLDL-ICs by
human macrophages. Our data also showed that nLDL-ICs
stimulated MAP kinase in a time-dependent manner. However, the concentration-dependent study (data not shown)
indicated that oxLDL-IC is more effective than nLDL-IC in
MAP kinase activation, suggesting that some components of
oxLDL may potentiate MAP kinase activation.
The most important result presented in this study is that
MAP kinase in THP-1 macrophage-like cells was activated
by human anti-oxLDL autoantibody-containing immune
complexes.26 Because it is not feasible to prepare insoluble
immune complexes with these antibodies, probably because
of the low affinity of these antibodies to oxLDL,4 we
developed a new model system in which the immune complexes were immobilized on type I collagen. The interaction
between oxLDL and collagen has been thoroughly studied in
past years.28,29 It has been well documented that a positivecharged collagenous domain in the macrophage scavenger
receptor is a critical binding site for negative-charged oxLDL.27 Furthermore, collagen is the major connective tissue
component of atherosclerotic arteries, constituting up to 40%
of the total protein in fibrous plaques and 60% in advance
lesions.40 Most of the collagen (50% to 75%) in a normal
artery or in a diseased intima is of type I.41,42 It has been
postulated that binding of oxLDL to collagens facilitates
accumulation of oxLDL in atherosclerotic lesions and uptake
of oxLDL by macrophages.43 In vitro studies showed that
oxLDL is bound to a greater extent to type I collagen gel than
to other types of collagens.28,29 In the present study, we
immobilized oxLDL on wells previously coated with type I
collagen gel, and then allowed human autoantibodies to bind
to the anchored oxLDL. THP-1 cells were then allowed to
interact with the anchored oxLDL-ICs by seeding the cells
into wells containing type I collagen-immobilized oxLDLICs. Our data show that immune complex formation is
required for MAP kinase activation because MAP kinase was
not stimulated when either autoantibodies or oxLDL was
absent in the collagen-based system. The involvement of
FcgRI in the stimulation was evidenced by the fact that IgG1
blocked the stimulation. These results suggest that immune
complexes prepared with purified human anti-oxLDL autoantibodies, like the insoluble immune complexes prepared
with rabbit anti-human LDL antibodies, stimulate MAP
kinase in human macrophages.
Activation of ERK MAP kinase in macrophages can be
triggered by a variety of extracellular stimuli, such as cytokines31, lipopolysaccharide,19 oxLDL,20 and oxLDL-IC as
described in the present study. Therefore, the upstream events
of MAP kinase activation elicited by these stimuli could be
different. Recently, studies have shown that MAP kinase can
be activated by oxidative stress induced by extracellular
stimuli.44,45 For example, it has been shown that angiotensin
II elicits a rapid increase in intracellular H2O2, which in turn
activates phosphorylation of p38 MAP kinase in vascular
1606
Arterioscler Thromb Vasc Biol.
July 1999
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smooth muscle cells.44 One of our previous studies showed
that LDL-IC stimulated a respiratory burst in human
monocyte-derived macrophages.11 Thus, it is possible that the
respiratory burst elicited by LDL-IC triggered the MAP
kinase activation in THP-1 macrophages. Further experiments are required to test this hypothesis.
Kusuhara et al20 have shown that pretreatment of smooth
muscle cells with PMA for 24 hours blocked oxLDL-induced
MAP kinase activation by .90%. Because prolonged stimulation of cells with PMA inhibits protein kinase C activity,
this result suggests that oxLDL stimulation of MAP kinase is
predominantly protein kinase C-dependent. In our present
study, we also found that oxLDL-IC failed to activate MAP
kinase when the stimulation was started immediately after
treatment of the cells with PMA for 24 hours (data not
shown). However, oxLDL-IC was able to activate MAP
kinase when the cells were washed after PMA treatment and
incubated with fresh culture medium for 24 hours before the
stimulation (Figure 1). These observations suggest that oxLDL-IC–induced MAP kinase activation is protein kinase
C-dependent.
In summary, this study demonstrated that oxLDL-ICs
stimulated MAP kinase activation in THP-1 macrophage-like
cells with or without CE loading and that the engagement of
FcgRI is required for the stimulation. The experiments using
type I collagen to immobilize oxLDL and to induce the
formation of anchored oxLDL-IC have shown for the first
time that human anti-oxLDL autoantibody-containing immune complexes stimulated MAP kinase. These data suggest
that oxLDL-ICs localized in atherosclerotic lesions may
interact with macrophage foam cells to stimulate the MAP
kinase signaling pathway, leading to macrophage activation.
Acknowledgments
This work was supported in part by grant HL-55782 from the
National Institutes of Health and a Grant-in-aid from the American
Heart Association (M.F.L.-V). We thank Dr Gabriel Virella for his
valuable advice. We also thank Charlyne Chassereau for the preparation of lipoproteins and Dr Victoria Velarde for her help in the
initial experiments.
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Oxidized LDL-Containing Immune Complexes Induce Fc Gamma Receptor I−Mediated
Mitogen-Activated Protein Kinase Activation in THP-1 Macrophages
Yan Huang, Ayad Jaffa, Sinikka Koskinen, Akira Takei and Maria F. Lopes-Virella
Arterioscler Thromb Vasc Biol. 1999;19:1600-1607
doi: 10.1161/01.ATV.19.7.1600
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