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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:990-999

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:990-999.)
© 1995 American Heart Association, Inc.

Articles

Immune Complexes of LDL Induce Atherogenic Responses in Human Monocytic Cells

Peter A. Kiener; Bruce M. Rankin; Patricia M. Davis; Sue A. Yocum; Glenn A. Warr; Robert I. Grove

From the Department of Autoimmunity and Transplantation, Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Wash (P.A.K., B.M.R., P.M.D., S.A.Y., R.I.G.), and the Department of Microbiology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Conn (G.A.W.).

Correspondence to Peter A. Kiener, Bristol-Myers Squibb Pharmaceutical Research Institute, 3005 First Ave, Seattle, WA 98121.


*    Abstract
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 *Abstract
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 down arrowResults
 down arrowDiscussion
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Abstract The ability of immune complexes of LDL or acetylated LDL (acLDL), together with antibodies to LDL, to induce a proatherogenic phenotype in human monocytic cells has been explored. Treatment of THP-1 monocytic cells or peripheral human monocytes with LDL immune complexes containing intact anti-LDL markedly enhanced the ability of these cells to subsequently bind and take up LDL, whereas aggregated LDL or LDL immune complexes prepared with F(ab')2 fragments of anti-LDL had no significant effect. Activation of THP-1 cells with intact LDL immune complexes also stimulated mRNA expression for the scavenger receptor. Additionally, activation of THP-1 cells with insoluble immune complexes of LDL or LDL stimulated generation of reactive oxygen intermediates that, in turn, could oxidize exogenous LDL. These results indicate that the binding of lipoprotein immune complexes to Fc receptors on monocytic cells activates a series of responses that could accelerate the initiation or progression of atherosclerosis.


Key Words: lipoproteins • scavenger receptors • Fc receptor • anti-LDL • TNF-{alpha}


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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There is accumulating evidence that immunologic mechanisms contribute to the development and progression of atherosclerosis (for reviews, see References 1 through 31 2 3 ). Macrophages and activated T lymphocytes are found in fatty streaks or atherosclerotic plaques,4 5 6 and complement becomes locally activated early in the deposition of fatty streaks.7 8 9 In animal models, circulating immune complexes (ICs) have been found to be potent proatherogenic factors,10 and autoimmune antibodies are reported to contribute to the pathogenesis of the disease.11 Because of these observations, it has been suggested that in humans, circulating ICs may predispose individuals to vascular disease.

It is thought that one of the critical steps in the development and progression of atherosclerosis is the formation of modified LDL (mLDL), leading to its enhanced uptake by the macrophage, which eventually results in the formation of lipid-laden foam cells. Development of these foam cells and their accumulation at subendothelial sites are key steps in the generation of atherosclerotic lesions.12 13 It has also recently been found that foam cells that are isolated from atherosclerotic lesions can themselves promote oxidation of LDL, thus perhaps contributing to the overall generation and uptake of mLDL.14 mLDL is taken up by the scavenger receptor on macrophages13 15 ; although this receptor may play a normal protective role in other tissues, it may be deleterious at the sites of plaque.16

More recently, additional mechanisms that lead to accumulation of cholesteryl esters in monocytes have been reported. ICs composed of LDL and antibodies to LDL can lead to enhanced LDL uptake and cholesteryl ester accumulation in human monocyte-derived macrophages. These ICs appear to be taken up by the Fc{gamma} receptor (Fc{gamma}R) system in monocytes/macrophages, either as free complexes or adsorbed to red blood cells.17 18 19 20 21 It was found that LDL ICs upregulated expression of the LDL receptor; however, the effect of these ICs on expression of the scavenger receptor and on the ability of the cells to subsequently take up mLDL through a non-Fc{gamma}R–mediated pathway was not reported.

Several studies have been carried out to determine whether antibodies to LDL or to mLDL do occur in vivo. Serum antibodies to oxidized LDL have been reported in patients with chronic periaortitis and in some healthy elderly subjects.22 A 30-fold greater level of anti-LDL has been found in patients with coronary atherosclerosis compared with that in healthy individuals.23 Autoantibodies to malondialdehyde-modified LDL have been found in the sera of both healthy subjects and those with coronary artery disease.24 25 Additionally, anti-mLDL antibodies, some present as ICs, have been found at the site of atherosclerotic lesions in both humans and rabbits.26

The role of ICs in the onset and progression of atherosclerotic disease is largely unexplored. It has been shown that ICs or immobilized antibodies to the Fc{gamma}R can stimulate production of a variety of cytokines3 27 28 29 30 31 and reactive oxygen intermediates.32 However, the effect of these substances on the oxidation of LDL, expression of the scavenger receptor, and hence, their role in the development of atherosclerotic lesions has not been well studied.

The monocytic cell line THP-1 expresses both Fc{gamma}RI and Fc{gamma}RII. It is possible to stimulate tyrosine phosphorylation, intracellular calcium mobilization, and cytokine production through these receptors.33 34 35 These cells have also been used as a model for studying the differentiation of monocytes into foam cells.36 37 38 When stimulated with phorbol 12-myristate 13-acetate (PMA), THP-1 cells undergo differentiation and take on macrophage-like characteristics. There is a marked increase in expression of the scavenger receptor, with a concomitant decrease in expression of the LDL receptor; additionally, upon incubation with acetylated LDL (acLDL), there is a significant increase in the levels of cholesteryl esters within the cell.37 These differentiated cells take up mLDL and take on the appearance of foam cells.

In this report we have evaluated the ability of lipoprotein-containing and nonlipoprotein-containing ICs to activate monocytic cells through the Fc{gamma}R. We show that both types of IC stimulate a cascade of events in the cells that would lead to an atherogenic phenotype, although IC-containing lipoproteins are significantly more effective. These results suggest that the formation and interaction of lipoprotein ICs with the Fc{gamma}R on monocytic cells could significantly contribute to oxidation of LDL and the generation of macrophage-derived foam cells, thus accelerating the onset or progression of atherosclerotic lesions.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
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 down arrowDiscussion
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Cells and Cell Lines
The human monocytic leukemia cell line THP-1 was obtained from the American Type Culture Collection and was cultured in RPMI 1640 medium supplemented with 10% (vol/vol) fetal calf serum (FCS), penicillin/streptomycin (100 U/mL and 100 µg/mL, respectively), gentamicin (20 µg/mL), and ß-mercaptoethanol (50 µmol/L). Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by density-gradient centrifugation in lymphocyte separation medium (Organon Teknika Corp). Purified monocytes were obtained from the PBMCs by "rosetting" the T cells with sheep red blood cells and then separating the remaining cells by centrifugal elutriation in RPMI 1640 containing 10 mmol/L HEPES and 1 µg/mL polymyxin B. Monocytes were determined to be >90% CD14+, as determined by fluorescence-activated cell sorting (FACS) analysis (data not shown).

Antibodies and Proteins
Anti-human LDL was either obtained from Biomedical Technologies Inc or prepared by immunization of rabbits with human LDL; both antisera recognized LDL and acLDL. To keep endotoxin levels to a minimum, the antisera were passed through a polymyxin B column (Pierce) prior to use. Goat anti-rabbit IgG, goat anti-mouse IgG, and goat anti-rat IgG were from Chemicon; mouse IgG2a, rabbit IgG, rat IgG, and lipopolysaccharide (LPS) were from Sigma Chemical Co. The monoclonal antibody that recognizes modified lysine groups on mLDL was a gift from Dr J.L. Witztum (University of California, San Diego) and has been described previously.39 The F(ab')2 fragment of rabbit polyclonal anti-LDL was prepared from the protein A–purified IgG fraction of the anti-LDL antisera by digestion with pepsin (0.005%, wt/wt) for 24 hours at 37°C. LDL was from Calbiochem; acLDL, 125I-acLDL (0.1 to 0.2 µCi/µg), and 1,1'-dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI)–acLDL were from Biomedical Technologies Inc. Aggregated LDL was prepared from LDL (10 mg/mL) by vortexing the solution for 5 minutes at room temperature. mLDL was prepared from LDL by derivatization with malonaldehyde bis(dimethyl acetal) as described previously.39 BSA (IgG-free, low endotoxin) was from Miles.

Preformed insoluble ICs of LDL or acLDL were prepared by titering the lipoproteins against anti-LDL antisera or the F(ab')2 fragment to give the maximum yield of precipitated complex. Similarly, nonlipoprotein ICs of goat anti-rabbit IgG, goat anti-mouse IgG, and goat anti-rat IgG were prepared by titering the goat antisera with the appropriate IgG. Complexes were formed in the presence of 5 µg/mL polymyxin B to keep endotoxin levels low. Following precipitation, the ICs were washed four times with cold PBS containing 0.05% NaN3. After the final wash, the precipitates were resuspended at 5 mg/mL in RPMI 1640 and stored at 4°C. The levels of endotoxin in the lipoproteins, antibodies, and ICs were determined with the QCL100 assay kit (M.A. Whittaker) and found to be <0.25 ng/mg protein or complex. The total protein concentration of the ICs was measured by using a Coomassie Brilliant Blue dye binding assay (Pierce) with soluble, human IgG as the protein standard. The levels of IgG (heavy chain) in the different ICs were estimated by dissolving the ICs in 2x sample buffer and separating several concentrations of each sample by SDS–polyacrylamide gel electrophoresis (SDS-PAGE). The samples were stained with Coomassie Brilliant Blue, and staining of the IgG heavy chain in each sample was quantitated by densitometric scanning.

Assay for H2O2
Induction and secretion of H2O2 in THP-1 cultures were determined by using the method of Ruch and coworkers.40 Cells were suspended at a density of 106 cells/mL in HBSS (phenol red–free) buffered with 25 mmol/L HEPES and supplemented with 0.1% (vol/vol) BSA, 25 µg/mL homovanillic acid, and 2 U/mL horseradish peroxidase (Sigma). After addition of the appropriate stimuli, the cultures were incubated for 1 hour at 37°C and the reactions were stopped by addition of 0.5 mL of 1 mol/L glycine, pH 11.0, containing 3 mmol/L EDTA. These samples were calibrated against a blank (cell-free substrate reaction mixture), and the fluorescence was determined (excitation wavelength, 312 nm; emission wavelength, 420 nm; slit, 5/5 nm) with a Perkin-Elmer LS-5 spectrofluorometer. All samples were analyzed in triplicate; H2O2 standards were prepared by using an extinction coefficient at 230 nm of 81 L/mol.

Assay for Oxidation of LDL by THP-1 Cells
Oxidation of LDL was determined as the amount of thiobarbituric acid–reacting substances (TBARS), using the method described by Morel and coworkers.41 THP-1 cells at a density of 1.5x106/mL were incubated with the appropriate stimulus in HBSS containing 100 µg/mL LDL at 37°C for 24 hours; controls without cells, for cell-free oxidation of LDL, were included in each series of assays. The cells were spun down and the cell-free medium was removed from each culture, treated with 20% (vol/vol) trichloroacetic acid, and heated with 1% (vol/vol) TBA for 45 minutes at 95°C. The samples were centrifuged at 12 000g for 30 seconds, the supernatants were removed, and their optical density at 532 nm was determined. Fresh solutions of TBA and malonaldehyde bis(dimethyl acetyl) standards (Aldrich Chemical Co) were prepared for each assay. Samples were assayed in triplicate.

Binding and Uptake of DiI-acLDL
THP-1 cells (2.0x105/mL) in media were treated with PMA, ICs, or other reagents in six-well plates for the indicated times. After the appropriate incubation time the cells were removed from the plates, the wells incubated twice with PBS containing 2.5 mmol/L EDTA, and the washes added back to the original cell suspensions. Elutriated monocytes (1.5x106/mL) were incubated with ICs in RPMI 1640 containing 20% (vol/vol) FCS in polypropylene tubes and then harvested by centrifugation. Both THP-1 cells and monocytes were spun down, washed once with 10 mL medium, resuspended in 1 mL medium containing 100 µg/mL LDL, and incubated for 15 minutes at 25°C. After this procedure the cells were spun down and resuspended in 1 mL medium containing 50 µg/mL LDL prior to the addition of 7.5 µg/mL diI-acLDL and, where appropriate, 500 µg/mL LDL or acLDL. Cells were incubated at 37°C for 4 hours, washed twice with 10 mL ice-cold PBS, and then fixed with PBS containing 0.5% (vol/vol) paraformaldehyde. The level of diI-acLDL bound and taken up by the cells was quantitated on a cell-by-cell basis, as described,36 42 43 on a FACScan analyzer with excitation at 488 nm and emission at 585 nm; 10 000 cells were counted for each sample, and gates were set to count only live cells. To normalize the results between different experiments, the data are presented as the mean fluorescence ratio, which is the mean fluorescence for uptake of diI-acLDL by treated cells divided by the mean fluorescence obtained for the uptake of diI-acLDL by untreated control cells in the same experiment.

Binding and Uptake of 125I-acLDL
THP-1 cells were incubated with ICs and prepared as outlined above. The binding and uptake of 125I-acLDL were measured under the same conditions as used for labelling with di-I acLDL, except that 2 to 3 µCi of 125I-acLDL was added to each sample. After 4 hours the cells were washed five times with PBS and then dissolved in 1 mL lysis buffer.34 The lysates were clarified by centrifugation at 13 000g for 10 minutes and the supernatants counted in an LKB gamma counter. All assays were done in triplicate.

Polymerase Chain Reaction (PCR) Analysis of THP-1 Cells
THP-1 cells were incubated with the ICs or PMA as described above. After harvest, the cells were lysed and the mRNA was isolated by using a Micro-FastTrack Isolation Kit (Invitrogen). Reverse transcriptase reactions (30 µL) using SuperScript RNaseH-Reverse Transcriptase (GIBCO BRL) were set up for each mRNA to yield the cDNA templates. PCRs using synthesized primer pair oligonucleotides were set up to amplify a 513-bp fragment (including restriction sites) of the scavenger receptor (Accession No. D90187: bp 34 to 520; sense strand, 5'GCG AAG CTT GAC GAA AGA AGT ATG GAG CAG TGG; antisense strand, 5' TCA GTC TCT AGA CTG CAG AAG AAT GTC ATT AAA TCT TTG) and a 231-bp fragment (including restriction sites) of the {gamma}-chain of the IgE receptor (Accession No. M33195: bp 80 to 283; sense strand, 5' GCT AGA GTC ACT AGT CTG GGA GAG CCT CAG CTC TGC; antisense strand, 5' GGT CGA TGG ATC CTG TGG TGG TTT CTC ATG CTT CAG). PCRs (50 µL) were carried out on a Perkin-Elmer 9600 thermocycler (30 or 45 cycles) using Pfu DNA polymerase (Stratagene). Analysis of the reverse transcriptase–PCRs was carried out on 1.2% agarose gels (SeaKem) in Tris acetate/EDTA buffer containing 1 µg/mL ethidium bromide. Gels were photographed under UV light. DNA fragment sizes were determined by comparison with DNA molecular-weight markers III and V (Boehringer Mannheim).

Tumor Necrosis Factor–{alpha} (TNF-{alpha}) Assays
THP-1 cells (106/mL) were incubated in RPMI 1640 with various concentrations of the ICs for 3 hours at 37°C, and the supernatants were removed and assayed by an enzyme-linked immunosorbent assay for TNF-{alpha} (Biosource) according to the manufacturer's instructions.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Fc{gamma}R Activation by ICs
To measure the ability of the various ICs to activate cells through the Fc{gamma}R and subsequently stimulate production of TNF-{alpha}, THP-1 cells were incubated with various concentrations of ICs, and the levels of TNF-{alpha} released into the cell supernatant 3 hours after activation were determined. Preliminary experiments had indicated that the TNF-{alpha} levels produced were found to be markedly affected by low levels of LPS in the IC, so polymyxin B (1 µg/mL) was included in all reactions. The preformed goat anti-rabbit IgG IC (nonlipoprotein containing) was consistently more effective at stimulating TNF-{alpha} production than was rabbit anti-LDL or acLDL IC (Fig 1ADown). ICs of F(ab')2 anti-LDL and LDL that could not bind to the Fc{gamma}Rs did not stimulate production of TNF-{alpha} (data not shown).



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  		Figure 1. Line plots showing activation of THP-1 cells with immune complexes (ICs). A, Dose-response curve of tumor necrosis factor–{alpha} production. Cells were stimulated with various concentrations of LDL (—{bullet}—) or rabbit IgG (--{circ}--) intact ICs (total protein) in RPMI 1640 at 37°C. After 3 hours 100 µL of the cell supernatant was removed and assayed for TNF-{alpha} by enzyme-linked immunosorbent assay. Assays were carried out in triplicate. Data (mean±SEM) are from five different experiments. B, Quantitation of IgG heavy chain in the ICs. Equivalent amounts (from the Coomassie Brilliant Blue protein determinations) of the different ICs were separated by SDS–polyacrylamide gel electrophoresis. Gels were stained, and the band in the gel corresponding to the IgG heavy chain was quantitated by densitometric scanning. —{bullet}— and --{circ}-- as in panel A. —{blacktriangleup}— indicates F(ab')2 anti-LDL/LDL IC. Data are from one experiment representative of a total of two.

To determine whether the differences in stimulation of TNF-{alpha} production could simply be attributed to the levels of IgG in the IC, the IgG heavy chain in the different complexes was quantitated. Equivalent amounts of total protein in the IC, over a range of protein concentrations, were separated by SDS-PAGE. The gels were stained with Coomassie Brilliant Blue, and staining of the band corresponding to the IgG heavy chain was scanned on a densitometer (Fig 1BUp). This analysis showed that the nonlipoprotein IC contained about two times more heavy chain than did the LDL IC containing intact rabbit anti-LDL. No IgG heavy chain was detected in the F(ab')2 anti-LDL IC by this method, indicating that there was <5% intact antibody in these complexes. Overall, the results showed that both types of IC can bind to Fc{gamma}Rs and stimulate cytokine production in monocytic cells; however, under the conditions used, the rabbit IgG (nonlipoprotein) ICs were more effective.

Binding and Uptake of DiI-acLDL
In atherosclerotic plaques, peripheral monocytes infiltrate and differentiate into macrophages that express the scavenger receptor; these are then able to take up large amounts of mLDL and eventually become foam cells. We determined whether stimulation of THP-1 cells with ICs would enhance the cell's ability to subsequently bind and take up acLDL. Fluorescence assays for measuring the binding and uptake of diI-acLDL through the scavenger receptor have been well documented.36 42 43

In unstimulated THP-1 cells, there was very little uptake of diI-acLDL (Fig 2Down). However 48 hours after treatment of the cells with 100 ng/mL PMA, which stimulates expression of the scavenger receptor, there was a marked increase in the uptake of the fluorescent label. Scavenger receptor expression reached a maximum 72 to 96 hours after stimulation with PMA, so in the initial experiments, measurements were taken 90 hours after addition of the IC.



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  		Figure 2. Fluorescence-activated cell sorting analysis of immune complex (IC) stimulation of binding and uptake of 1,1'-dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI)–acetylated (ac) LDL in THP-1 cells. THP-1 cells were treated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) or 100 µg/mL of intact ICs of LDL or rabbit IgG for 90 hours at 37°C in RPMI 1640 containing 10% fetal calf serum. The cells were harvested, washed, and then incubated with diI-acLDL for 4 hours at 37°C. Binding and uptake of diI-acLDL were then measured as described in "Methods." Data are from one experiment; similar data were obtained in five separate experiments.

Stimulation of THP-1 cells with nonlipoprotein IC (rabbit or rat IgG, 200 µg/mL) enhanced uptake of acLDL to a small but significant degree (Figs 2Up and 3Down). However, activation of cells with ICs containing intact anti-LDL together with LDL or acLDL enhanced uptake of the fluorescent label to a much greater degree (Fig 2Up). As shown in the FACS profiles, stimulation of THP-1 cells with either PMA or lipoprotein IC gave rise to a very heterogeneous population of cells that took up acLDL to varying degrees. On occasion, treatment of THP-1 cells with LDL IC gave rise to an apparently biphasic response, with one population of cells taking up higher levels of diI-acLDL than the other. However, this was not observed consistently. There was little uptake of diI-acLDL into cells stimulated with LPS (2 µg/mL) or acLDL (Fig 3Down) and LDL (25 µg/mL) or anti-LDL (10 to 25 µg/mL) (data not shown).



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  		Figure 3. Bar graph of role of Fc receptors in the activation of THP-1 cells. THP-1 cells were incubated with immune complexes (ICs) of rabbit or rat IgG (200 µg/mL), acetylated (ac) LDL (25 µg/mL), lipopolysaccharide (LPS, 2 µg/mL), aggregated (agg) LDL (50 µg/mL), LDL containing intact anti-LDL (LDL IC 50 µg/mL), LDL containing F(ab')2 anti-LDL (50 µg/mL), or phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) for 90 hours at 37°C in RPMI 1640 containing 10% fetal calf serum. The cells were harvested and washed, and then the binding and uptake of 1,1'-dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine perchlorate-acLDL for 4 hours at 37°C were measured by fluorescence-activated cell sorting analysis as described in "Methods." Data (mean±SEM) are compiled from six different experiments.

Because the various ICs that contained intact IgG were able to activate both intracellular signaling and cytokine production in monocytic cells through the Fc{gamma}Rs, we thought it likely that stimulation of scavenger receptor expression was also mediated through these receptors. To confirm this, the ability of aggregated LDL or an LDL IC that contained F(ab')2 anti-LDL to induce uptake of acLDL was determined. As indicated earlier, treatment of THP-1 cells for 90 hours with 50 µg/mL of an LDL IC (containing intact anti-LDL) very effectively increased the uptake of diI-acLDL (Fig 3Up). Very little increase in uptake of diI-acLDL was seen in cells treated with either a F(ab')2 LDL IC (50 µg/mL) or aggregated LDL (50 µg/mL). This finding indicates that binding of the complexes to Fc{gamma}Rs appears to be necessary for upregulation of expression of the acLDL receptor.

To characterize the induction of scavenger receptor expression in more detail, the dose response of the cells to the various ICs was followed. THP-1 cells were incubated with different concentrations of complexes for 90 hours and the uptake of diI-acLDL was subsequently determined (Fig 4ADown). At all concentrations of IC, those containing intact anti-LDL and lipoproteins were markedly more effective at stimulating uptake of the diI-acLDL than were those without lipoprotein.



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  		Figure 4. Dose response and kinetics of immune complex (IC) stimulation of uptake of 1,1'-dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine perchlorate–acetylated (ac) LDL in THP-1 cells. A, Dose-response curves for uptake of diI-acLDL. THP-1 cells were stimulated with various concentrations of LDL (—{bullet}—) or rabbit IgG (--{circ}--) intact IC (total protein) for 90 hours at 37°C in RPMI 1640 containing 10% fetal calf serum. The cells were harvested and washed, and then the binding and uptake of diI-acLDL for 4 hours at 37°C were measured by fluorescence-activated cell sorting (FACS) analysis as described in "Methods." Data are plotted as mean fluorescence ratios (±SEM) compiled from five different experiments. B, Kinetics line plot for stimulation of uptake of diI-acLDL. THP-1 cells were stimulated with 50 µg/mL intact IC (total protein) at 37°C in RPMI 1640 containing 10% fetal calf serum. At various times the cells were harvested and washed, and the uptake of diI-acLDL over 4 hours at 37°C was determined by FACS analysis. —{bullet}— and --{circ}-- as in panel A. Results shown are plotted as mean fluorescence ratios (±SEM) from five different experiments.

To determine the kinetics of cell-surface expression of the scavenger receptor, THP-1 cells were stimulated with 50 µg/mL IC for various times. The cells were harvested and washed, as outlined ealier, and their ability to bind and take up diI-acLDL was measured. Enhanced uptake of diI-acLDL was observed within 24 hours of activation of the cells with ICs containing LDL. This ability to take up acLDL increased over the next 5 days (Fig 4BUp). In cells stimulated with nonlipoprotein ICs, an increase in uptake of diI-acLDL was detectable 24 to 48 hours following activation of the cells, and this, too, increased slightly over the next 5 days. Throughout the time course of these experiments, uptake of diI-acLDL by cells that had been treated with nonlipoprotein IC was higher than the very low uptake by unstimulated cells but markedly less than that of cells treated with complexes containing lipoproteins and intact anti-LDL.

Uptake of diI-acLDL presumably occurred through the scavenger receptor. However, it was necessary to show that uptake was indeed through this receptor rather than either through the LDL receptor or by the formation of additional ICs that could be taken up through the Fc{gamma}R. To do this, cells were stimulated with the appropriate complexes for 90 hours, the media were removed, and the cells were washed and, where appropriate, excess LDL or acLDL was added back to the samples prior to incubation with the fluorescent label. acLDL at 500 µg/mL markedly inhibited uptake of diI-acLDL by cells pretreated with PMA or lipoprotein IC, whereas LDL at the same concentration had very little effect (Fig 5Down). These results indicate that uptake was mediated through a receptor specific for acLDL.



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  		Figure 5. Bar graphs showing effect of excess LDL or acetylated (Ac) LDL on the uptake of 1,1'-dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine perchlorate–acLDL by THP-1 cells. Cells were incubated for 90 hours at 37°C in RPMI 1640 containing 10% fetal calf serum with A, phorbol 12-myristate 13-acetate (100 ng/mL); B, rabbit IgG immune complex (IC) (100 µg/mL); C, LDL IC (50 µg/mL); and D, acLDL IC (50 µg/mL). The cells were harvested, washed, and then incubated with diI-acLDL in the presence of no further additions, 500 µg/mL soluble acLDL, or 500 µg/ml LDL for 4 hours at 37°C. Uptake of diI-acLDL was then assessed by fluorescence-activated cell sorting analysis as described in "Methods." Data are mean fluorescence ratios (±SEM) from six different experiments.

Binding and Uptake of 125I-acLDL
Very similar results were obtained when cells were activated with ICs and the uptake of 125I-acLDL was measured. Stimulation of THP-1 cells with LDL IC enhanced uptake of 125I-acLDL, whereas nonlipoprotein ICs were less effective (Fig 6ADown). Similar treatment of THP-1 cells with PMA markedly enhanced the ability of the cells to take up the radiolabel (Fig 6BDown). Following either mode of cell activation, acLDL very significantly blocked uptake of the label, whereas LDL was much less effective.



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  		Figure 6. Bar graphs showing immune complex (IC) stimulation of binding and uptake of 125I acetylated (ac) LDL by THP-1 cells. THP-1 cells were treated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) or 50 µg/mL intact ICs of LDL or rabbit IgG (nonlipoprotein) for 90 hours. The cells were harvested, washed, and then incubated with 125I-acLDL at 37°C for 4 hours. Incorporation of 125I-acLDL in the presence or absence of excess soluble LDL was measured as described in "Methods." Assays were carried out in triplicate. A, Uptake of 125I-acLDL following stimulation with ICs; B, uptake of 125I-acLDL following stimulation with PMA. Data are plotted as counts per minute (cpm) and are from one representative experiment of three.

Activation of Peripheral Monocytes
The effect of ICs on the ability of monocytes to bind and take up acLDL was also determined. Monocytes were isolated by elutriation and then incubated with LDL intact IC or nonlipoprotein (rabbit IgG) complexes. Within 16 hours of isolation, the untreated (control) monocytes in culture were themselves able to take up significant levels of diI-acLDL. This was decreased by incubation with the nonlipoprotein (rabbit IgG) IC; however, treatment of the cells with the LDL IC markedly increased (approximately twofold to threefold) the ability of monocytes to take up diI-acLDL (Fig 7ADown and 7BDown). After prolonged incubation of the elutriated monocytes in culture (>50 hours, the unstimulated monocytes took up very high levels of diI-acLDL; this appeared to be partially inhibited by both types of IC (data not shown).




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  		Figure 7. Immune complex (IC) stimulation of binding and uptake of 1,1'-dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine perchlorate–acetylated (ac) LDL by peripheral human monocytes. A, Fluorescence-activated cell sorting (FACS) profile of elutriated monocytes, not stimulated (control) or stimulated with 100 µg/mL rabbit IgG IC or 100 µg/mL LDL intact IC. Elutriated monocytes were cultured for 16 hours at 37°C in RPMI 1640 containing 20% fetal calf serum. B, Mean fluorescence ratio data of cells. Samples were harvested and washed, and then the binding and uptake of diI-acLDL for 4 hours at 37°C were measured by FACS analysis as described in "Methods." Data (mean±SEM) are compiled from elutriated monocytes from seven different donors.

PCR Analysis of Scavenger Receptor Expression
The previous experiments indicated that activation of THP-1 cells with intact ICs containing either LDL or acLDL markedly enhanced binding and uptake of acLDL by stimulating expression of the scavenger receptor. To determine whether an increase in the mRNA for the scavenger receptor could be detected following stimulation with ICs, mRNA was isolated from THP-1 cells and analyzed by PCR. In each experiment, all samples from control or activated cells were isolated, analyzed by PCR, and run on gels at the same time. The data shown (Fig 8Down) are representative of three different experiments, and PCR analyses in all three gave very similar results. These experiments showed that there was either no or low expression of a band corresponding to the scavenger receptor mRNA in control cells (lane 1). However, within 48 hours of stimulation of the cells with either 100 ng/mL PMA (lane 5) or 100 µg/mL LDL intact IC (lane 3), a marked increase in the levels of message could be detected. Expression of mRNA for the {gamma}-chain of the lg{epsilon} receptor was used as a control in these experiments and did not significantly change following stimulation of THP-1 cells with PMA or ICs (lanes 2, 4, and 6). Although these results are only semiquantitative, they do confirm that both PMA and lipoprotein ICs can stimulate an increase in mRNA expression for the scavenger receptor.



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  		Figure 8. Polymerase chain reaction (PCR) analysis of mRNA from THP-1 cells. THP-1 cells were left untreated (control) or activated by treatment with phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) or LDL immune complex (IC) (100 µg/mL) for 48 hours at 37°C in RPMI 1640 containing 10% fetal calf serum. mRNA isolation and PCR analysis were carried out as described in "Methods." Lanes 1, 3, and 5 show reaction with primers for the scavenger receptor; lanes 2, 4, and 6, the reaction with primers for the Fc{epsilon} {gamma} chain (band runs at 231 bp). Samples were run on an agarose gel, and bands were visualized by staining with ethidium bromide and photographed. Arrow indicates fragment of the scavenger receptor DNA, which runs as a band of 513 bp. PCRs and analysis of samples from control cell or cells treated with ICs were run in the same reaction and analyzed on the same gels. Data shown are one experiment representative of three different experiments.

Stimulation of Oxidation by LDL IC
Our results indicate that LDL IC could stimulate signal-transduction pathways in monocytic cells that lead to an increase in expression of the scavenger receptor. It has previously been shown that activation of monocytes through the Fc{gamma}R can stimulate production of reactive oxygen intermediates.31 To determine whether activation of THP-1 cells by ICs could lead to modification of LDL, the effect of various ICs on H2O2 production and oxidation of LDL was determined. Within 1 hour, the different insoluble ICs all strongly stimulated production of H2O2 in THP-1 cells (Fig 9ADown). Soluble complexes at saturating concentrations were much less effective but produced levels of H2O2 that were elevated over those from unstimulated cells (Fig 9BDown). LPS alone was without effect and also did not significantly alter the levels of H2O2 produced by the LDL IC (Fig 9BDown). Antibodies alone, or LDL and acLDL alone, stimulated very little production.



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  		Figure 9. Bar graphs showing stimulation of production of H2O2 and oxidation of LDL in THP-1 cells. A and B, Production of H2O2. Cells at 106/mL were stimulated for 1 hour at 37°C in HBSS containing 25 mmol/L HEPES, 0.1% BSA, 25 µg/mL homovanillic acid, and 2 U/mL horseradish peroxidase, together with 100 µg/mL of the various insoluble immune complexes (IC), or 25 µg/mL soluble LDL or acetylated (ac) LDL (with, where appropriate, 10 µg/mL anti-LDL); zymosan was used at 100 µg/mL. Assays were done in triplicate as described in "Methods"; data are from one representative experiment of three. C, Oxidation of LDL. Total oxidation of exogenous, soluble LDL over 24 hours was measured following incubation of the cells (1.5x106/mL) at 37°C for 24 hours in HBSS containing 100 µg/mL LDL together with the appropriate stimulus. The cells were spun down, and the supernatants were removed and treated with trichloroacetic acid and thiobarbituric acid as described in "Methods." Samples were assayed in triplicate. Results shown are one experiment from a total of two. MDA indicates malondialdehyde; rab, rabbit.

To follow whether the IC-induced reactive oxygen species in the cells were sufficient to stimulate oxidation of exogenous soluble LDL, THP-1 cells were incubated with 100 µg/mL of the various ICs for 24 hours at 37°C in the presence of 100 µg/mL soluble LDL. Oxidation of soluble LDL in the medium over this 24-hour incubation was assessed by measuring the generation of TBARS in the cell supernatants, as described in "Methods." All of the insoluble, intact ICs markedly stimulated oxidation of LDL by the cells (Fig 9CUp). Soluble ICs also stimulated oxidation, but even at saturating concentrations, the levels were only about 25% to 30% of that observed with the insoluble ICs. No significant oxidation of LDL over that seen with unstimulated cells was caused by incubating the cells with LPS, antibodies, or lipoproteins alone.


*    Discussion
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 *Discussion
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Development of atherosclerotic plaques arises from a complicated network of both cell-cell and cell-protein interactions. It is thought that the immune system actively contributes to both the onset and progression of atherosclerosis,1 2 but the role of the various cells, antibodies, lymphokines, cytokines, and other circulating proteins is poorly understood (reviewed in References 3 and 443 44 ).

During the early stages of atherogenesis, circulating monocytes bind to and penetrate the endothelium; they then take up and metabolize lipoproteins and eventually form fatty streaks. Although the mechanisms for the uptake of lipoproteins are not clear, it is thought that the scavenger receptors on these macrophages play a major role in the internalization of oxidized or mLDL. ICs of LDL and anti-LDL may also provide an additional pathway for uptake of lipoproteins through the surface Fc{gamma}R.17 18 19 20 During plaque development, T cells and macrophages become activated,4 45 but the significance of this event remains unclear. These cells infiltrate into the lesion site and, as the lesion progresses, the smooth muscle cells (SMCs) proliferate and deposit large amounts of tissue matrix,3 4 5 6 eventually forming fibrous plaques. These plaques contain SMCs, T cells, and macrophages.

The monocytic cell line THP-1 has been used as a model for the differentiation of circulating monocytes into macrophage-derived foam cells.35 36 Freshly isolated elutriated monocytes (before being placed in culture) have relatively low levels of scavenger receptor; upon culture, this level markedly increases. In this study, we used THP-1 cells to investigate the effects of ICs on the expression of the scavenger receptor. Stimulation of THP-1 cells with insoluble ICs and, to a lesser degree, soluble ICs, activated production of H2O2 and stimulated oxidation of LDL. Additionally, ICs that contained intact IgG stimulated both THP-1 cells and peripheral monocytes to bind and take up acLDL. Excess acLDL but not LDL was able to inhibit the uptake of labeled acLDL, indicating that uptake was specific for a receptor that bound acLDL but that did not recognize LDL. This finding also argues against uptake of the fluorescent diI-acLDL through the Fc{gamma}R as an anti-LDL/diI-acLDL complex. The antibodies used in these experiments recognize both LDL and acLDL, so that if an antibody on the cell surface was available to bind additional antigen, both excess LDL and excess acLDL would have blocked uptake of diI-acLDL. PCR analysis of cells stimulated with ICs confirmed the increase in expression of the scavenger receptor. Together, these results indicate that acLDL (scavenger) receptor expression is induced by stimulation of cells with ICs. Preliminary data have also shown that activation of cells with insoluble ICs containing lipoproteins produce cell supernatants that are mitogenic to vascular SMCs (data not shown).

Although both types of intact IC (with or without lipoproteins) stimulated expression of the scavenger receptor and uptake of acLDL with similar kinetics in THP-1 cells, the LDL ICs were significantly more effective over a 20-fold concentration range. The differences in ability of the ICs to stimulate scavenger receptor expression cannot be explained by the nonlipoprotein ICs being less able to bind and activate THP-1 cells, as these ICs very effectively stimulated production of H2O2 and production of TNF-{alpha} through the Fc{gamma}R. Additionally, estimation of the IgG heavy-chain content of the ICs revealed that there was two to three times more IgG in the nonlipoprotein ICs than in the LDL ICs. Cell activation does require uptake of the IC through Fc{gamma}R, as the LDL complexes composed of F(ab')2 anti-LDL did not stimulate the cells. While it is difficult to assess the relative affinities of the different ICs for the Fc{gamma}Rs, it is clear that although rabbit IgG and rat IgG IC can effectively bind to and stimulate some functions of the monocyte, they are less effective at stimulating expression of the scavenger receptor. Additionally, at present it is not clear which FcR is stimulated by the various ICs to give rise to expression of the scavenger receptor.

Stimulation of peripheral monocytes with lipoprotein ICs also showed a marked increase in the ability of cells to take up acLDL within 16 hours. However, after prolonged incubation, elutriated monocytes, when kept in culture, spontaneously expressed very high levels of scavenger receptor. Nonlipoprotein ICs appeared to partially inhibit this enhanced uptake. The reason for this is not known at present.

Our studies suggest that incubation of THP-1 cells or human peripheral monocytes with ICs containing LDL or acLDL may activate through the Fc{gamma}R a cascade of proatherogenic responses. Although nonlipoprotein ICs may contribute to atherogenesis,10 11 it is clear that the presence of lipoproteins in the IC could very significantly contribute to both the generation of oxidized LDL and stimulation of its uptake into cells by enhancing expression of the scavenger receptor. In vivo, these responses would result in accelerated formation of atherosclerotic plaques by increasing the formation and uptake of mLDL by monocytes/macrophages. Insoluble ICs were much more effective stimulators of the various responses than were soluble ICs. Immobilization of ICs may occur in vivo, as circulating antibodies to LDL or mLDL either form complexes with LDL aggregates or come into contact with deposits of LDL or its derivatives in the vessel wall. The presence of ICs of antibodies with epitopes to oxidized LDL and mLDL have been found in both human and rabbit atherosclerotic lesions.26

Insoluble ICs were much more effective than soluble ICs in stimulating production of TNF-{alpha}, releasing reactive oxygen intermediates, and enhancing uptake of acLDL. In agreement with our results, it has previously been observed that immobilized antibodies stimulate production of cytokines much more efficiently than do soluble antibodies.29 30 The nonlipoprotein ICs stimulated production of H2O2, but this was less than that produced by the LDL IC. Both treatments, however, gave rise to similar levels of oxidation of exogenous, soluble LDL. The reasons for this are not clear but may simply indicate that with the LDL IC, oxidation of insoluble LDL in the complexes may also occur. This would not be detected in our assays, during which we followed the oxidation of soluble LDL in the medium.

Our results differ from those reported by Morganelli and coworkers,46 who used bispecific anti-LDL and anti-Fc{gamma}R antibodies. The bispecific antibody complexes were taken up through the Fc{gamma}R and resulted in degradation of LDL, but unlike the results reported in our study, this did not give rise to production of significant levels of reactive oxygen intermediates, oxidation of LDL, or induction of the acLDL receptor. The major difference between the two studies is that we used insoluble ICs, whereas Morganelli and coworkers used soluble ICs. As discussed above, our data show that soluble ICs are much less able to activate monocytic cells, a finding similar to that reported by Gisinger et al.20

It has been reported that internalization of the LDL IC increases uptake and metabolism of LDL and over 20 hours upregulates expression of the LDL receptor.21 The effect of the IC on scavenger receptor expression and oxidation of LDL was not determined. We are currently studying the effect of ICs on expression of the LDL receptor by following more prolonged incubations. Preliminary experiments indicate that stimulation of THP-1 cells with LDL ICs for 48 hours significantly reduces expression of the LDL receptor (data not shown).

Finally, we found that stimulation of THP-1 cells with ICs gave rise to the production of TNF-{alpha} and of reactive oxygen intermediates. Recently, it has been reported that LDL ICs can stimulate release of cytokines.31 It should be noted that TNF-{alpha} would enhance adhesion of monocytes to the endothelium,47 48 and we have shown here that reactive oxygen species can oxidize LDL. LPS, which also induces monocytes to produce TNF-{alpha}, did not significantly stimulate expression of the scavenger receptor; we did not evaluate its ability to inhibit IC-induced expression of the scavenger receptor. We found that production of TNF-{alpha} and of reactive oxygen intermediates was enhanced by priming the cells with interferon-{gamma} (data not shown), which may be produced by activated T cells in the plaques.6 These responses could contribute further to the generation of plaque. Conversely, it has been reported that TNF-{alpha} (induced by LPS) and interferon {gamma} can inhibit spontaneous expression of the scavenger receptor in human monocytes.49 50 In our experiments, even though the LDL IC stimulated production of TNF-{alpha}, this was insufficient to prevent upregulation of the scavenger receptor in THP-1 cells. Stimulation of peripheral monocytes with the LDL IC induced a twofold to threefold increase in the levels of scavenger receptor within 16 hours, but prolonged treatment with the IC (>50 hours) partially inhibited the spontaneous increase that occurred in untreated monocytes. This decrease after longer treatment could be due to production of TNF-{alpha}. We are currently studying this. It is clear that the physiological events that occur in the microenvironment of the lesion will critically influence plaque development.

In summary, our work has demonstrated that stimulation of the THP-1 monocytic cell line or of peripheral monocytes through the Fc{gamma}R with lipoprotein ICs can dramatically increase the proatherogenic response. These include stimulation of the oxidation of LDL, induction of the scavenger receptor for acLDL, and production of TNF-{alpha}. Combined, these responses would be expected to accelerate progression of atherosclerosis in the vessel wall.


*    Acknowledgments
 
We would like to thank Dr J.L. Witztum of the La Jolla Specialized Center of Research on Atherosclerosis (University of California, San Diego) for helpful discussions and for his gift of anti-mLDL monoclonal antibody.

Received September 19, 1994; accepted May 5, 1995.


*    References
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 *References
 

  1. Hansson GK, Jonasson I, Seifert PS, Stemme S. Immune mechanisms in atherosclerosis. Arteriosclerosis. 1989;9:567-578. [Abstract/Free Full Text]
  2. Parums, DV. Inflammatory mediators in atherosclerosis. Biochem Soc Trans. 1990;18:1069-1072. [Medline] [Order article via Infotrieve]
  3. Ross, R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809. [Medline] [Order article via Infotrieve]
  4. Jonasson L, Holue T, Skalli O, Bondjers G, Hansson G. Regional accumulation of T cells, macrophages and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis. 1986;6:131-138. [Abstract/Free Full Text]
  5. Mitchinson MJ, Ball RY. Macrophages and atherogenesis. Lancet. 1987;2:146-148. [Medline] [Order article via Infotrieve]
  6. Parums DV, Ramshaw AL. Immunohistochemical characterization of inflammatory cells associated with advanced atherosclerosis. Histopathology. 1990;17:543-552. [Medline] [Order article via Infotrieve]
  7. Seifert PS, Hugo F, Hansson G, Bhakdi S. Prelesional complement activation in experimental atherosclerosis. Lab Invest. 1989;60:747-754. [Medline] [Order article via Infotrieve]
  8. Vlaicu R, Niculescu F, Rus HG, Cristea A. Immunohistochemical localization of the terminal C5b-9 complement complex in human aortic fibrous placque. Atherosclerosis. 1985;57:163-177. [Medline] [Order article via Infotrieve]
  9. Pang AS, Katz A, Minta JO. C3 deposition in cholesterol-induced atherosclerosis in rabbits: a possible etiologic role for complement in atherogenesis. J Immunol. 1979;123:1117-1123. [Abstract/Free Full Text]
  10. Minick CR, Murphy GE. Experimental induction of atheroarteriosclerosis by the synergy of allergic injury to arteries and lipid-rich diet, II: effect of repeatedly injected foreign proteins in rabbits fed a lipid-rich, cholesterol-poor diet. Am J Pathol. 1973;73:265-300. [Medline] [Order article via Infotrieve]
  11. Cerelli J, Brasile L, Karmody A. Role of the vascular endothelial cell antigen system in the etiology of atherosclerosis. Ann Surg. 1985;202:329-334. [Medline] [Order article via Infotrieve]
  12. Gerrity RG. The role of the monocyte in atherogenesis, I: transition of blood borne monocytes into foam cells in fatty lesions. Am J Pathol. 1981;103:181-190. [Abstract]
  13. Goldstein JL, Ho YK, Basu SK, Brown MS. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc Natl Acad Sci U S A. 1979;76:333-337. [Abstract/Free Full Text]
  14. Rosenfeld ME, Khoo JC, Miller E, Parthasarathy S, Palinski W, Witztum JL. Macrophage-derived foam cells freshly isolated from rabbit atherosclerotic lesions degrade modified lipoproteins, promote oxidation of low density lipoproteins and contain oxidation-specific protein specific adducts. J Clin Invest. 1991;87:90-99.
  15. Fogelman AM, Schecter I, Seager J, Hokom M, Child JS, Edwards PA. Malondialdehyde alteration of LDL leads to cholesteryl ester accumulation in human monocyte-macrophages. Proc Natl Acad Sci U S A. 1980;77:2214-2218. [Abstract/Free Full Text]
  16. Krieger M. Molecular flypaper and atherosclerosis: structure of the macrophage scavenger receptor. Trends Biochem Sci. 1992;17:141-146. [Medline] [Order article via Infotrieve]
  17. Klimov AN, Denisenko AD, Popov AV, Nagornev VA, Pleskov VM, Vinogradov AG, Denisenko TV, Magracheva EY, Kheifes GM, Kuznetzov AS. Lipoprotein-antibody immune complexes: their catabolism and role in foam cell formation. Atherosclerosis. 1985;58:1-15. [Medline] [Order article via Infotrieve]
  18. Khoo JC, Miller E, Pio F, Steinberg D, and Witztum JL. Monoclonal antibodies against LDL further enhance macrophage uptake of LDL aggregates. Arterioscler Thromb. 1992;12:1258-1266. [Abstract/Free Full Text]
  19. Griffith RL, Virella GT, Stevenson HC, Lopes-Virella MF. LDL metabolism by human macrophages activated with LDL immune complexes. J Exp Med. 1988;168:1041-1059. [Abstract/Free Full Text]
  20. Gisinger C, Virella GT, Lopes-Virella MF. Erythrocyte bound LDL immune complexes lead to cholesteryl ester accumulation in human monocyte derived macrophages. Clin Immunol Immunopathol. 1991;59:37-52. [Medline] [Order article via Infotrieve]
  21. Lopes-Virella MF, Griffith RL, Shunk KA, Virella GT. Enhanced uptake and impaired intracellular metabolism of LDL complexed with anti-LDL antibodies. Arterioscler Thromb. 1991;11:1356-1367. [Abstract/Free Full Text]
  22. Parums DV, Brown DL, Mitchinson MJ. Serum antibodies to oxidized LDL and ceroid in chronic periaortitis. Arch Pathol Lab Med. 1990;114:383-387. [Medline] [Order article via Infotrieve]
  23. Orekhov AN, Tertov VV, Kabakov AE, Adamova IY, Pokrovsky SN, Smirnov VN. Autoantibodies against modified LDL. Arterioscler Thromb. 1991;11:316-326. [Abstract/Free Full Text]
  24. Salonen JT, Yla-Herttuala S, Yamamoto R, Butler S, Korpela H, Salonen R, Nyyssonen K, Palinski W, Witztum JL. Autoantibody against oxidized LDL and progression of carotid atherosclerosis. Lancet. 1992;339:883-887. [Medline] [Order article via Infotrieve]
  25. Palinski W, Rosenfeld ME, Yla-Herttuala S, Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum JL. Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci U S A. 1989;86:1372-1376. [Abstract/Free Full Text]
  26. Yla-Herttuala S, Palinski W, Butler SW, Picard S, Steinberg D, Witztum JL. Rabbit and human atherosclerotic lesions contain IgG that recognize epitopes of oxidized LDL. Arterioscler Thromb. 1994;14:32-40. [Abstract/Free Full Text]
  27. Ben-Sasson SZ, LeGros G, Conrad DH, Finkelman FD, Paul WE. Cross-linking Fc receptors stimulates splenic non-B, non-T cells to secrete interleukin 4 and other lymphokines. Proc Natl Acad Sci U S A. 1990;87:1421-1425. [Abstract/Free Full Text]
  28. Krutmann J, Kirnbauer R, Kock A, Schwarz T, Schopf E, May LT, Sehgal PB, Luger TA. Cross-linking Fc receptors on monocytes triggers IL-6 production: role in anti-CD3-induced T cell activation. J Immunol. 1990;145:1337-1342. [Abstract]
  29. Debets JMH, van der Linden CJ, Dieteren IEM, Leeuwenberg JFM, Buurman WA. Fc receptor cross-linking induces rapid secretion of tumor necrosis factor (cachectin) by human peripheral blood monocytes. J Immunol. 1988;141:1197-1201. [Abstract]
  30. Debets JMH, Van de Winkel JGJ, Ceuppens JL, Dieteren IEM, Buurman WA. Cross-linking of both Fc-gammaRI and Fc-gammaRII induces secretion of tumor necrosis factor by human monocytes, requiring high affinity Fc-Fc-gammaR interactions. J Immunol. 1990;144:1304-1310. [Abstract]
  31. Lopes-Virella MF, Virella G. Atherosclerosis and autoimmunity. Clin Immunol Immunopathol. 1994;73:155-167. [Medline] [Order article via Infotrieve]
  32. Yamamato K, Johnson RB. Dissociation of phagocytosis from stimulation of the oxidative metabolic burst in macrophages. J Exp Med. 1984;159:405-416. [Abstract/Free Full Text]
  33. Liao F, Shin HS, Rhee SG. Tyrosine phosphorylation of phospholipase C-gamma I induced by cross-linking of the high-affinity or low-affinity Fc receptor for IgG In U937 cells. Proc Natl Acad Sci U S A. 1992;89:3659-3663. [Abstract/Free Full Text]
  34. Rankin BM, Yocum SA, Mittler RS, Kiener PA. Stimulation of tyrosine phosphorylation and calcium mobilization by Fc-{gamma} receptor crosslinking: regulation by the phosphotyrosine phosphatase CD45. J Immunol. 1993;150:605-616. [Abstract]
  35. Scholl PR, Ahern D, Geha RS. Protein tyrosine phosphorylation induced via the IgG receptor Fc-{gamma}RI and Fc-{gamma}RII in the human monocytic cell line THP-1. J Immunol. 1992;148:1751-1757.
  36. Via DP, Pons L, Dennison DK, Fanslow AE, Bernini F. Induction of acetyl-LDL receptor activity by phorbol ester in the human monocyte cell line THP-1. J Lipid Res. 1989;30:1515-1524. [Abstract]
  37. Banka CL, Black AS, Dyer CA, Curtiss LK. THP-1 cells form foam cells in response to co-culture with lipoproteins but not platelets. J Lipid Res. 1991;32:35-43. [Abstract]
  38. Moulton KS, Wu H, Barnett J, Parthasarathy S, Glass CK. Regulated expression of the human acetylated LDL receptor gene and isolation of promoter sequences. Proc Natl Acad Sci U S A. 1992;89:8102-8106. [Abstract/Free Full Text]
  39. Palinski W, Yla-Herttuala S, Rosenfield ME, Butler SW, Socher SA, Parthasarathy S, Curtiss LK, Witztum JL. Antisera and monoclonal antibodies specific fo epitopes generated during oxidative modification of low density lipoprotein. Arteriosclerosis. 1990;10:325-335. [Abstract/Free Full Text]
  40. Ruch W, Cooper PH, Baglioni M. Assay of H2O2 production by macrophages and neutrophils with homovanillic acid and horseradish peroxidase. J Immunol Methods. 1983;63:347-357. [Medline] [Order article via Infotrieve]
  41. Morel DW, Hessler JR, Chisolm GM. LDL cytotoxicity induced by free radical peroxidation of lipid. J Lipid Res. 1983;24:1070-1076. [Abstract]
  42. Akeson AL, Schroeder K, Woods C, Schmidt CJ, Jones WD. Suppression of IL1ß and LDL scavenger receptor expression in macrophages by a selective protein kinase C inhibitor. J Lipid Res. 1991;32:1699-1707. [Abstract]
  43. Suzuki K, Sakata N, Kitani A, Hara M, Hirose T, Hirose W, Norioka K, Harigai M, Kawagoe M, Nakamura H. Characterization of human monocytic cell line, U937, in taking up acetylated LDL and cholesteryl ester accumulation: a flow cytometric and HPLC study. Biochim Biophys Acta. 1990;1042:210-216. [Medline] [Order article via Infotrieve]
  44. Libby P, Hansson GK. Involvement of the immune system in human atherogenesis: current knowledge and unanswered questions. Lab Invest. 1991;64:5-15. [Medline] [Order article via Infotrieve]
  45. Hansson GK, Jonasson L, Lojsthed B, Stemme S, Kocher O, Gabbiani G. Localization of T lymphocytes and macrophages in fibrous and complicated human atherosclerotic plaques. Atherosclerosis. 1988;72:135-141. [Medline] [Order article via Infotrieve]
  46. Morganelli PM, Kitzmiller TJ, Hemmer R, Fanger MW. Redirected targeting of LDL to human monocyte Fc{gamma} receptors with bispecific antibodies. Arterioscler Thromb. 1992;12:1131-1138. [Abstract]
  47. Bevilacqua MP, Pober JS, Wheeler ME, Cotran RS, Gimbrone MA. IL-1 acts on cultured human vascular endothelium to increase their adhesion on polymorphonuclear leukocytes, monocytes and related leukocyte cell lines. J Clin Invest. 1985;76:2003-2011.
  48. Pober JS, Gimbrone MA, Lapierre DL, Mendrick DL, Rothlein R, Springer TA. Overlapping patterns of activation of human endothelial cell by IL-1, TNF and immune interferon. J Immunol. 1986;137:1893-1896. [Abstract]
  49. Geng YJ, Hansson GK. Interferon {gamma} inhibits scavenger receptor expression and foam cell formation in human monocyte derived macrophages. J Clin Invest. 1992;89:1322-1330.
  50. van Lenten BJ, Fogelman AM. LPS-induced inhibition of scavenger receptor expression in human monocyte-macrophages is mediated through TNF-{alpha}. J Immunol. 1992;148:112-116.[Abstract]



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