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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1600-1607.)
© 1999 American Heart Association, Inc.

Vascular Biology

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

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 virellam{at}musc.edu

	Abstract

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Abstract—Our previous studies have shown that Fc gamma receptor (FcR)-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 FcR triggers activation of signal transduction pathways that alter gene expression in macrophages. In this study, we determined whether engagement of FcR 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 FcR 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.

Key Words: oxidized LDL • immune complex • mitogen-activated protein kinase • Fc gamma receptor

	Introduction

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In 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 2 3 4 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 oxLDL-ICs 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 (FcRI) led to the transformation of macrophages into foam cells^{7 8 9 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 (TNF) and interleukin-1ß (IL-1ß), and enhanced respiratory burst.¹¹ It has been shown that TNF and IL-1ß have a considerable impact in the development of atherosclerosis.^{12 13 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 FcRI by LDL-IC.⁸ It has been documented that cross-linking of FcRI triggers signal transduction pathways that modulate macrophage functions.^{15 16 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- expression.^{19 20 21} These observations prompted us to investigate whether engagement of FcRI 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-loading of the cells induced a more sustained MAP kinase activation. Engagement of FcRI by oxLDL-ICs was required for the activation of MAP kinase because blocking of FcRI 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 FcRI-mediated MAP kinase activation in THP-1 macrophage-like cells.

	Methods

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Materials
Rabbit anti-phosphorylated and anti-p42/p44 MAP kinase antibodies were purchased from New England Biolabs, Inc. Anti-phosphotyrosine 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 µmol/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 µmol/L EDTA, pH 8.0, passed through an Acrodisc filter (0.22-µm 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, Cu²⁺ was added at a final concentration of 10 µmol/L to the LDL preparation and the mixture incubated at 37°C for 18 hours. To stop the oxidation reaction 200 µmol/L EDTA and 40 µmol/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 µg/mL) or native LDL (170 µg/mL) with a rabbit anti-LDL antiserum (500 µg/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-µg 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 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. ß-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 -70°C. Before the performance of the FcRI 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 FcRI with high affinity and does not bind to any of the other FcR 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 macrophage-like 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 µmol/L CuCl₂. To isolate 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 µg/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 µg/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 (2.7x10⁶/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.

	Results

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Time-Dependent Stimulation of MAP Kinase in THP-1 Macrophage-Like Cells by OxLDL-ICs
THP-1 macrophage-like cells were incubated with 150 µg/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.

View larger version (28K): [in this window] [in a new window]   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 µg/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.

Concentration-Dependent Stimulation of MAP Kinase by OxLDL-ICs
THP-1 macrophage-like cells were incubated with 0, 25, 50, 100, 150, or 200 µg/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 µg/mL of oxLDL-IC. Higher levels of oxLDL-IC had a similar effect. Therefore, we selected 150 µg/mL of oxLDL-IC as the optimal concentration to stimulate cells in the subsequent experiments.

View larger version (46K): [in this window] [in a new window]   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.

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 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 µg/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.

View larger version (27K): [in this window] [in a new window]   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 µg/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 mean±SD of 3 experiments run in triplicate.

View larger version (34K): [in this window] [in a new window]   Figure 4. Time course of MAP kinase phosphorylation in CE-laden THP-1 macrophage-like cells stimulated with oxLDL-ICs. THP-1 macrophage-like cells were treated with 100 µg/mL of acetylated LDL to induce CE accumulation as described above. The cells were then stimulated with 150 µg/mL of oxLDL-ICs for the periods indicated. Twenty-five micrograms of cellular proteins was subjected to immunoblotting using anti-phosphorylated 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.

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 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).

View larger version (88K): [in this window] [in a new window]   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 µg/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.

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.

View larger version (28K): [in this window] [in a new window]   Figure 6. Nuclear translocation of MAP kinase in oxLDL-IC–stimulated THP-1 macrophage-like cells. THP-1 macrophage-like cells were incubated in SFM or SFM supplemented with 150 µg/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 anti-phosphorylated 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.

Engagement of Type I FcR by OxLDL-ICs Is Required for MAP Kinase Phosphorylation
We have previously shown that LDL-ICs are taken up by THP-1 cells through FcRI.⁸ To determine whether engagement of FcRI 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 FcRI, or anti-CD32 monoclonal antibody (IV3), which binds to type II FcR. The results showed that human IgG1 blocked MAP kinase phosphorylation in a dose-dependent manner with 90% inhibition at 10 µg/mL, whereas anti-CD32 monoclonal antibodies had no effect (Figure 7). These data suggest that engagement of FcRI 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 FcRI in THP-1 macrophages,⁸ these data further confirm that the engagement of FcRI is required for MAP kinase activation.

View larger version (41K): [in this window] [in a new window]   Figure 7. Blocking of oxLDL-IC–stimulated MAP kinase phosphorylation by monomeric human IgG1. THP-1 macrophage-like cells were incubated with 150 µg/mL of oxLDL-ICs for 30 minutes in the presence of increasing concentrations of monomeric human IgG1 or of anti-FcRII (CD32) monoclonal antibodies (IV3) as indicated. The cells were lysed after the incubation, and 25 µg of cell proteins was used for Western blot analysis of MAP kinase phosphorylation as described in Methods.

View larger version (27K): [in this window] [in a new window]   Figure 8. Stimulation of MAP kinase by immune complexes containing native LDL. THP-1 macrophage-like cells were incubated with 150 µg/mL of native LDL-containing immune complexes for 0, 5, 10, 20, or 30 minutes and then lysed. Twenty-five 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.

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 phosphorylation 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).

View larger version (80K): [in this window] [in a new window]   Figure 9. Stimulation of MAP kinase by immune complexes containing human anti-oxLDL autoantibodies. OxLDL (100 µg/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 µg/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 (2.7x106/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).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The role of oxLDL-IC in atherogenesis has been the subject of intensive investigation by our and other research groups.^{1 2 3 4 5 6 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, and smooth muscle cell proliferation.^{12 13 14 30} We have recently shown that LDL-ICs preferentially engaged FcRI in human macrophages and that this engagement of FcRI is required for activation of these cells.⁸ Thus, it is likely that LDL-IC–induced macrophage activation is mediated by FcRI-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 TNF-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 FcRI by oxLDL-IC is required to induce oxLDL-IC–mediated MAP kinase activation. It has been shown that cross-linking of FcRI 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 FcRI-dependent 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 FcRI with high affinity, and an anti-CD32 monoclonal antibody (IV3),⁸ to block the binding of FcRI and FcRII, respectively, by oxLDL-IC. These monomeric antibodies are unable to cross-link FcRs 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 anti-CD32 had no effect, suggesting that the activation of MAP kinase by oxLDL-IC is FcRI-dependent. These results are consistent with our previous finding⁸ that FcRI 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 positive-charged 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 oxLDL-ICs. 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 FcRI 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 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 FcRI 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.

Received August 3, 1998; accepted December 11, 1998.

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