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Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:106-114

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:106-114.)
© 1996 American Heart Association, Inc.

Articles

Decrease in Scavenger Receptor Expression in Human Monocyte–Derived Macrophages Treated With Granulocyte Macrophage Colony-Stimulating Factor

Maaike A. van der Kooij; Olivier H. Morand; Herman J. Kempen; Theo J.C. van Berkel

From the Pharma Division (M.A. van der K., O.H.M., H.J.K.), Preclinical Research, F Hoffmann–La Roche Ltd, Basel, Switzerland, and the Division of Biopharmaceutics (M.A. van der K., T.J.C. van B.), Leiden/Amsterdam Center for Drug Research, University of Leiden, Leiden, Netherlands.

Correspondence to Maaike A. van der Kooij, Pharma Division, Preclinical Research, PRPV, B68/340, F Hoffmann–La Roche Ltd, CH-4002 Basel, Switzerland.


*    Abstract
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Abstract To determine whether scavenger receptors are susceptible to regulation by granulocyte macrophage colony-stimulating factor (GM-CSF), a macrophage-specific cytokine, human monocytes were differentiated into macrophages in the absence or presence of 20 U/mL GM-CSF. Binding, uptake, and degradation of acetylated LDL (Ac-LDL) and oxidized LDL (Ox-LDL) were measured. Treatment with GM-CSF resulted in a significant twofold to threefold decrease in the number of binding sites for Ac-LDL and Ox-LDL on the surface of macrophages without affecting the affinity of the receptor for these ligands. Competition experiments revealed that two binding sites were responsible for the recognition and uptake of Ac-LDL: one specific for Ac-LDL and one that recognized both Ac-LDL and Ox-LDL. No binding site specific for Ox-LDL could be detected in either control or GM-CSF–treated macrophages. Treatment of human monocyte–derived macrophages with GM-CSF resulted in a decrease of the Ac-LDL/Ox-LDL receptor but did not affect the binding site specific for Ac-LDL. Northern blot analysis showed that mRNA levels of both types I and II scavenger receptor were reduced in macrophages differentiated in the presence of GM-CSF. Human macrophages that were differentiated in the presence of GM-CSF accumulated {approx}50% fewer cholesteryl esters. Taken together, these results indicate that GM-CSF can downregulate both types I and II scavenger receptor in human monocyte–derived macrophages, which might have implications for foam cell formation.


Key Words: granulocyte macrophage colony-stimulating factor • atherosclerosis • macrophage • scavenger receptor • cholesterol


*    Introduction
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 up arrowAbstract
 *Introduction
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down arrowDiscussion
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Scavenger receptors that are expressed on the cell surface during the differentiation of monocytes into macrophages mediate the uptake of modified lipoproteins such as Ac-LDL, Ox-LDL, and maleylated albumin as well as certain polynucleotides, some polysaccharides, bacterial LPSs, and other macromolecules.1 2 3 4 5 In contrast to the LDL receptor, scavenger receptors are not regulated by the cholesterol content of the cell. Degradation of modified lipoproteins taken up via the scavenger receptor pathway can result in massive cholesterol accumulation and cholesterol esterification in macrophages that ultimately lead to the transformation of macrophages into foam cells.1 6 7 Macrophage-derived foam cells are an important feature of early atherosclerotic lesions,8 9 and it is thought that the development of lipid-laden foam cells in the atherosclerotic plaque is an important early event in the process leading to atherosclerosis. However, the regulation of scavenger receptors in mature macrophages is not fully understood.

Cytokines play a critical role in the development of atherosclerosis. Cytokine mRNA levels are elevated in atherosclerotic lesions.10 11 12 Cytokines can be secreted by a variety of cells, including endothelial cells, smooth muscle cells, lymphocytes, and monocyte-macrophages.13 14 15 Among cytokines, M-CSF and GM-CSF more specifically regulate macrophage activity. Besides their role in proliferation and differentiation of monocytic progenitor cells,16 17 they also stimulate functions of mature macrophages, such as tumoricidal activity, chemotaxis, phagocytosis, and cytotoxicity.18 19 20 M-CSF can modulate scavenger receptor expression in vitro,21 22 but information on the effect of GM-CSF on scavenger receptor expression is scarce.

In this study, human monocytes were treated with GM-CSF during differentiation into macrophages, and scavenger receptor mRNA levels as well as scavenger receptor activity by means of cellular association and degradation of 125I-Ac-LDL and 125I-Ox-LDL and the ability for cholesterol loading were examined. The data show that GM-CSF triggers downregulation of both scavenger receptor types I and II in human macrophages.


*    Methods
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 *Methods
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Materials
M-199, penicillin, streptomycin, PBS, and pyruvate were purchased from GIBCO BRL (Life Technologies Ltd). Ficoll-Paque was from Pharmacia Biotech. Sodium [125I]iodide (carrier free in 0.1 mol/L NaOH), Hybond-N membranes, and [32P]dCTP were purchased from Amersham International. Human recombinant GM-CSF was from Boehringer Mannheim. The Quantikine Immunoassay for human GM-CSF was purchased from R&D Systems. The Gene Amp RNA PCR kit was from Perkin Elmer Cetus. Human liver poly A+ RNA was obtained from Clontech. The pCR II vector was from Invitrogen. Escherichia coli XL-1 blue strain, the Sequenase version 2.0 DNA sequencing kit, and the Prime-it II random primer labeling kit were from Stratagen. Human serum for mixed lymphocyte cultures (H-2520), human recombinant M-CSF, poly(I), HSA (fraction V), and the {alpha}-naphthyl butyrate esterase staining kit were purchased from Sigma Chemical Co. BCA reagent was from Pierce. All other reagents and solvents of the highest purity available were purchased from Fluka or Sigma.

Isolation of Human LDL, Iodination, and Chemical Modification
Plasma from the blood of healthy volunteers was obtained at the Blutspendezentrum in Basel, collected in EDTA-containing tubes, and stored at -20°C. LDL was isolated by sequential ultracentrifugation at 1.019<d<1.063 g/mL.23 Prior to chemical modification, LDL was iodinated with 125I at a specific activity of 160 to 350 cpm/ng apolipoprotein by using the iodine monochloride procedure of McFarlane24 as modified by Bilheimer et al.25 LDL and 125I-LDL were acetylated by repeated additions of acetic anhydride26 followed by extensive dialysis against 150 mmol/L NaCl containing 0.3 mmol/L EDTA, pH 7.4. For oxidation of LDL, the concentration of EDTA in the lipoprotein preparation was first reduced by dialyzing it against PBS containing 10 µmol/L EDTA at 4°C. Next the oxidation of LDL or 125I-LDL was performed by exposing 200 µg apolipoprotein/mL to 10 µmol/L free Cu2+ at 37°C.27 After 20 hours the oxidation was stopped by complexing the Cu2+ with 0.2 mmol/L EDTA. Ox-LDL was dialyzed against at least two changes of 2 L PBS containing 0.2 mmol/L EDTA, pH 7.4, at 4°C overnight. The change in electrophoretic mobility was checked on an agarose gel by using the Paragon lipoprotein kit (Beckman). Migration of chemically modified lipoproteins was compared with that of unmodified LDL and expressed as relative electrophoretic mobility. The electophoretic mobility relative to LDL was 2.25±0.14 (n=11) and 2.66±0.15 (n=8) for Ac-LDL and Ox-LDL, respectively. All lipoprotein preparations were sterilized by a 0.45-µm membrane filtration, stored at 4°C in the presence of 40 µmol/L t-butylated-4-hydroxytoluene, and used within 2 weeks.

Preparation and Culture of Macrophages
Fresh EDTA-treated buffy coats from the blood of healthy volunteers were obtained at the Blutspendezentrum in Basel and diluted with 20 mL PBS–0.2% bovine serum albumin. Mononuclear cells were isolated by a Ficoll-Paque centrifugation.28 Briefly, 5 mL of diluted buffy coat was layered over 10 mL Ficoll-Paque and centrifuged at 500g for 30 minutes at room temperature. The mixed mononuclear cell fraction and serum were removed by aspiration, and cells were washed with PBS–0.2% bovine serum albumin. The Ficoll-Paque centrifugation was repeated once to remove the remaining erythrocytes. The repurified mononuclear cell fraction was washed three times with PBS–0.2% bovine serum albumin. Finally, the cells were resuspended in M-199 containing 10% human serum, 100 U/mL penicillin, 100 mg/mL streptomycin, and 1 mmol/L pyruvate and seeded in 48-well plates (Costar) at a density of 5x105 monocytes per well. After 2 to 3 hours of incubation at 37°C in 5% CO2 and 95% air, nonadherent cells were removed by three washes with serum-free M-199. Medium was replaced by fresh M-199 containing 10% human serum and antibiotics with or without 20 U/mL GM-CSF. Monocyte-derived macrophages were used within 7 to 11 days after seeding, and are referred to as human macrophages as judged by cell morphology and nonspecifc esterase staining of the cells. The medium of control cells was checked for GM-CSF content by using the quantitative sandwich enzyme immunoassay of R&D Systems, which has a detection limit of 2 pg/mL.

Binding of 125I-Ac-LDL and 125I-Ox-LDL to Human Macrophages
Binding of 125I-Ac-LDL and 125I-Ox-LDL to human macrophages was determined after incubation with the radiolabeled ligand at concentrations ranging from 0 to 40 µg/mL in HEPES-buffered M-199–2% HSA at 4°C for 4 hours. Cells were then washed with cold PBS–0.2% HSA and incubated on ice for another 20 minutes. After three additional washes with HSA-free PBS, cells were solubilized with BCA protein reagent, pH 11, microwaved (150 W) for 1 minute, and incubated at 37°C for 30 minutes. By using this procedure the radioactivity was directly and completely solubilized in the protein reagent. Aliquots were taken for protein measurement at 562 nm in a microplate reader as well as for counting of radioactivity. Nonspecific binding was defined as the binding of the radiolabeled ligand in the presence of a 30-fold and 10-fold excess of unlabeled Ac-LDL and Ox-LDL, respectively. Binding data were fitted according to the equation y=Mx/(K+x)+sx, where x is the concentration of the ligand, y is bound radioactivity, M is maximum specific binding capacity (Bmax), K is binding affinity (Kd), and s is constant. The data shown are representative of two or more experiments.

Cell Association and Degradation of 125I-Ac-LDL and 125I-Ox-LDL
To determine the cell association and degradation of Ac-LDL and Ox-LDL, macrophages were incubated with different concentrations of radiolabeled Ac-LDL and Ox-LDL in M-199–2% HSA at 37°C for 4 hours. Cell-associated radioactivity was measured as described above, and degradation was assessed by measuring the trichloroacetic acid–soluble, nonchloroform-extractable radioactivity in the medium.29 Nonspecific association and degradation were assessed in the presence of 200 µg/mL poly(I) for 125I-Ac-LDL and 1 mg/mL unlabeled Ox-LDL for 125I-Ox-LDL.

In competition experiments, 125I-Ac-LDL or 125I-Ox-LDL (both 5 µg/mL) were incubated with increasing concentrations of different competitors in M-199–2% HSA at 37°C for 4 hours. Nonspecific association and degradation were defined as the association and degradation of the radiolabeled ligand measured in the presence of 400 µg/mL unlabeled ligand. Control incubations were performed in wells containing no cells, and the values were subtracted from all experimental values.

Oligonucleotide Probe Preparation
An anti-sense oligonucleotide was constructed on the basis of the cDNA sequence for the human scavenger receptor.30 The sequence of the selected oligonucleotide corresponded to bases 328 through 662 of exon IV, a sequence that encodes for the {alpha}-helical coiled-coil domain of the scavenger receptor protein and is shared by both types I and II receptor. The two nucleotide sequences of sense and anti-sense primers were 5'-CAAAGTCTCACGGGAAAAGG-3' and 5'-AGGTATTCTCTTGGATTTTG-3', respectively (MWG-Biotech), and were designed by using OLIGO software, version 4.0. Human liver RNA poly A+ was first reverse transcribed by using the Gene Amp RNA PCR kit before the oligonucleotide was synthesized and amplified in a DNA synthesizer (Perkin Elmer Cetus DNA thermal cycler). Twenty-five PCR cycles were performed, each consisting of a sequence of 60 seconds at 94°C, 60 seconds at 45°C, and 60 seconds at 60°C. The PCR product detected by ethidium bromide staining was found to migrate between the 300- and 400-bp marker in a 3.5% agarose gel.

To check whether the sequence of the PCR product was correct, the oligonucleotides were cloned into pCR II, transformed in E coli XL-1 blue strain, and selected for LacZ gene sensitivity after growing the strain on LB-Amp plates overnight. Twenty-four colonies were isolated from 400 positive colonies, and plasmids were restricted by using EcoRI and checked for the presence of the PCR product. Seven subcolonies contained the product. Two of these (subcolonies 5 and 10) were sequenced by using the M13 reverse primer, the M13(-40) forward primer, and the two primers used with the PCR. Subcolony 5 was 100% complementary with bases 328 through 662, the domain selected on exon IV of the human scavenger receptor gene. The probe was labeled with [32P]dCTP and used for the detection of human scavenger receptor mRNA.

RNA Isolation
All glassware and solutions were treated with diethylpyrocarbonate to inhibit RNases. Total RNA was isolated from adherent human macrophages cultured in the presence or absence of GM-CSF according to the method of Chomczynski and Sacchi.31 Briefly, the cells were lysed in a denaturing buffer containing 4 mol/L guanidinium-thiocyanate, 25 mmol/L sodium citrate, 0.5% lauryl sarcosine, and 0.1 mol/L 2-mercaptoethanol, pH 7.0. RNA was extracted by mixing the cell lysate with an equal volume of phenol, 0.1 vol 2 mol/L sodium acetate, pH 4.0, and 0.2 vol chloroform–isoamyl alcohol (49:1). After centrifugation at 12 000 rpm at 4°C for 20 minutes, the upper phase was collected, and RNA was precipitated at -20°C for at least 1 hour in an equal volume of isopropanol. The pellet obtained after centrifugation at 12 000 rpm at 4°C for 20 minutes was washed in 70% ethanol. After another centrifugation the RNA pellet was dissolved and was quantified by spectrophotometry at 260 nm.

Northern Blot Analysis
Total RNA prepared from control cells and GM-CSF–treated cells (10 µg per lane) was fractionated by electrophoresis through a denaturing formaldehyde 1% agarose gel. RNA was transferred to Hybond-N membranes by electroblotting. After covalent linkage of the transferred RNA by UV irradiation for 5 minutes, membranes were prehybridized for 2 hours at 42°C in 8 mL prehybridization mix containing 50% formamide, 5x saline–sodium citrate buffer, 50 µg/mL sheared salmon sperm DNA, 0.1% SDS, 0.1% Ficoll, and 0.1% polyvinylpyrolidone. Hybridization with the 32P-labeled oligonucleotide probe was done in the presence of 10% dextran sulfate in prehybridization mix in a Hybaid oven overnight. Membranes were washed with a final stringency of 0.2x saline–sodium citrate containing 0.1% SDS at 50°C. The membranes were exposed to Hyperfilm-ECR films (Amersham), and bands were quantified by using a Phosphoimager (Molecular Dynamics). RNA blots were reprobed with a 32P-labeled S18 probe to ensure equal RNA loading.

Cholesterol Loading in Macrophages
Adherent human macrophages were washed three times with 0.5 mL serum-free M-199 medium. Cells were then incubated with 0.5 mL M-199 containing 2% human LPDS with or without Ac-LDL or Ox-LDL (100 µg/mL each) in the absence or presence of 20 U/mL GM-CSF for 4 days. At the end of the incubation, cells were washed three times with PBS containing Ca2+ and Mg2+. Cellular lipids were extracted from the wells according to the method of Hara and Radin.32 Lipids were dissolved in isopropanol, and total and unesterified cholesterol were quantified by using a fluorimetric enzymatic method with p-hydroxyphenylacetic acid as fluorochrome.33 Pure free cholesterol and cholesteryl oleate were used as standards. The protein content in each well was determined directly after isopropanol extraction by using the BCA reagent.


*    Results
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 up arrowMethods
*Results
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Effect of GM-CSF on Proliferation and Differentiation of Macrophages
Fig 1ADown and 1BDown show phase-contrast photomicrographs of human macrophages differentiated in the absence or presence of GM-CSF for 7 days. Under both conditions macrophages increased in size and exhibited the typical "fried egg" cell morphology. No detectable amount of GM-CSF was found in the medium of three different control macrophage preparations. The contamination of standard macrophage preparations by lymphocytes was less than 2% as judged by nonspecific esterase staining (Fig 1CDown and 1DDown).



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  		Figure 1. Cell morphology of human monocyte–derived macrophages differentiated in the absence or presence of 20 U/mL human recombinant GM-CSF for 7 days. Cells were fixed in 3% paraformaldehyde containing 60 mmol/L saccharose in PBS at room temperature for 10 minutes, after which they were either directly embedded in DePeX mounting medium or stained for {alpha}-naphthyl butyrate esterase activity and then embedded in DePeX. A and B, Phase-contrast photomicrographs of control (A) and GM-CSF–treated (B) macrophages; C and D, nonspecific esterase staining of control (C) and GM-CSF–treated (D) macrophages (magnification x400).

After 1 day of differentiation the cellular protein content per well for GM-CSF–treated macrophages was already twice that of control macrophages (Table 1Down). The cellular protein content per well for control macrophages decreased by 30% between days 1 and 7. In contrast, the cellular protein content per well for GM-CSF–treated macrophages increased threefold during the same time. A likely explanation for these data is that GM-CSF can act as a growth stimulus for monocyte-macrophages.16 17 GM-CSF could also prevent detachment of macrophages during the 7 days of differentiation.


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  		Table 1. Effect of GM-CSF on Cellular Protein Content per Well

Binding of 125I-Ac-LDL and 125I-Ox-LDL to Control and GM-CSF–Treated Macrophages
The expression of scavenger receptors on human macrophages can be modulated by cytokines such as interferon gamma,34 35 transforming growth factor–ß1,36 and M-CSF.22 To determine whether scavenger receptors of the mature macrophage are susceptible to regulation by GM-CSF, human macrophages were cultured in the absence or presence of GM-CSF (20 U/mL) for 7 days, and the binding of increasing concentrations of 125I-Ac-LDL and 125I-Ox-LDL was examined at 4°C (Fig 2Down). 125I-Ac-LDL and 125I-Ox-LDL exhibited saturable, high-affinity binding to the macrophages. Upon GM-CSF treatment, the maximal binding capacity of 125I-Ac-LDL and 125I-Ox-LDL was reduced to 35% and 30% of the control values, respectively. Computer analyses of the binding data showed that the reduction in 125I-lipoprotein binding was due to a decrease in the number of binding sites on the cell surface and not to a change in affinity of the ligands for their receptors, as illustrated by Scatchard plots (Fig 2Down) and summarized in Table 2Down. The apparent Kd of 125I-Ac-LDL and 125I-Ox-LDL for control macrophages was 3.9 and 6.8 µg/mL, respectively, ie, not significantly different from the Kd calculated for macrophages treated with GM-CSF (5.5 and 7.4 µg/mL, respectively). In contrast, Bmax was reduced about threefold for both Ac-LDL and Ox-LDL in GM-CSF–treated cells compared with control cells. These data indicate that the reduction in binding observed for 125I-Ac-LDL and 125I-Ox-LDL is mainly caused by a reduction in the number of receptors expressed on the surface of macrophages.



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  		Figure 2. Saturation curves and Scatchard plots for specific binding of 125I-Ac-LDL and 125I-Ox-LDL to human monocyte–derived macrophages differentiated in the absence ({circ}) or presence ({bullet}) of 20 U/mL GM-CSF for 7 days. Binding was measured at 4°C for 4 hours as described in "Methods." Specific binding was calculated by subtracting nonspecific binding from total binding. Data are expressed in nanograms protein per milligram cellular protein. Each point represents the mean±SEM of three different experiments. B indicates bound; F, free.


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  		Table 2. Apparent Binding Parameters of 125I-Ac-LDL and 125I-Ox-LDL

Cell Association and Degradation of Radiolabeled Ac-LDL and Ox-LDL in Control and GM-CSF–Treated Macrophages
To determine the effect of GM-CSF on the uptake and degradation efficiency of modified lipoproteins, control human macrophages and GM-CSF–treated macrophages were incubated at 37°C with increasing concentrations of 125I-Ac-LDL and 125I-Ox-LDL. After 4 hours, cell-associated radioactivity as well as the trichloroacetic acid–soluble, nonchloroform-extractable radioactivity in the medium was measured. Cell association of 125I-Ac-LDL and 125I-Ox-LDL was saturable in macrophages differentiated in the absence and presence of GM-CSF (Fig 3ADown and 3BDown). Maximum specific association of 125I-Ox-LDL was slightly higher than that of 125I-Ac-LDL. Treatment of macrophages with GM-CSF resulted in a decrease of specific association for both 125I-Ac-LDL and 125I-Ox-LDL. Degradation of Ac-LDL and Ox-LDL was also saturable with increasing concentrations of radiolabeled modified lipoprotein (Fig 3CDown and 3DDown). In contrast to Ac-LDL, degradation of Ox-LDL by macrophages was unchanged upon treatment with GM-CSF.



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  		Figure 3. Saturation curves for cell association with and degradation of 125I-Ac-LDL and 125I-Ox-LDL to human monocyte–derived macrophages differentiated in the absence ({circ}) or presence ({bullet}) of 20 U/mL GM-CSF. Specific association of 125I-Ac-LDL and 125I-Ox-LDL was measured at 37°C for 4 hours as described in "Methods" and corrected by subtracting cell association obtained in the presence of 200 µg/mL poly(I) and 1 mg/mL unlabeled Ox-LDL, respectively. Specific degradation of 125I-Ac-LDL and 125I-Ox-LDL was corrected similarly. Each point represents the mean±SEM of three (A and B) or two (C and D) different experiments.

In similar experiments differentiating macrophages were treated with 128 ng/mL M-CSF for 7 days. M-CSF increased cell association and degradation of 125I-Ox-LDL consistently with previous reports (data not shown).21 22

To analyze the specificity of the recognition sites mediating the interaction of Ac-LDL and Ox-LDL with macrophages, control cells and GM-CSF–treated cells were incubated with radiolabeled Ac-LDL or Ox-LDL at 5 µg/mL at 37°C together with increasing concentrations of unlabeled Ac-LDL, Ox-LDL, LDL, and poly(I) for 4 hours (Fig 4Down). No effect of the competitors on protein content per well was observed up to a concentration of 400 µg/mL. Unlabeled Ac-LDL effectively blocked the degradation of 125I-Ac-LDL; at a concentration of 400 µg/mL, more than 90% of the degradation was blocked (Fig 4ADown). Poly(I) was also very effective in competing with 125I-Ac-LDL degradation. LDL was ineffective, while Ox-LDL at concentrations up to 400 µg/mL blocked Ac-LDL degradation to a maximum of 50%. In GM-CSF–treated macrophages Ox-LDL could also compete with 125I-Ac-LDL degradation to a maximum of 25% (Fig 4BDown). Unlabeled Ac-LDL, Ox-LDL, and poly(I) completely blocked 125I-Ox-LDL degradation, whereas LDL had no effect (Fig 4CDown). Similar results were obtained when GM-CSF–treated macrophages were tested (Fig 4DDown). Taken together, these data suggest that Ac-LDL is recognized both by an Ac-LDL–specific binding site and an Ac-LDL/Ox-LDL binding site. Ox-LDL interacts only with the Ac-LDL/Ox-LDL binding site. No site specific for Ox-LDL could be detected in either control or GM-CSF–treated macrophages.



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  		Figure 4. Effect of increasing concentrations of LDL ({circ}), Ac-LDL ({square}), Ox-LDL ({blacksquare}), and poly(I) ({bullet}) on total degradation of 125I-Ac-LDL (A and B) or 125I-Ox-LDL (C and D) by human monocyte–derived macrophages differentiated in the absence or presence of 20 U/mL GM-CSF for 7 days. Nonspecific degradation of 125I-Ac-LDL and 125I-Ox-LDL was 2% to 5% and 20% to 25% of total degradation, respectively. Control values for 125I-Ac-LDL were 5489±670 and 3365±1706 ng/mg cell protein for untreated and GM-CSF–treated macrophages, respectively; these values for 125I-Ox-LDL were 920±374 and 1082±558 ng/mg cell protein. Each point represents the mean±SEM of three different experiments.

Selective Effects of GM-CSF on Scavenger Receptor Subtypes
Absolute values of association and degradation of Ac-LDL and Ox-LDL at 5 µg/mL of ligand contributed by the two receptors were calculated (Table 3Down). Cell association and degradation of Ac-LDL mediated by the Ac-LDL–specific binding site did not change upon GM-CSF treatment. In contrast, differentiation of macrophages in the presence of GM-CSF led to a twofold to threefold reduction of both association and degradation mediated by the Ac-LDL/Ox-LDL binding site. Cell association and degradation decreased from 109 to 45 and 3180 to 1009 ng lipoprotein/mg protein, respectively. The specific cell association of Ox-LDL, which is also mediated by the Ac-LDL/Ox-LDL binding site, was consistently reduced twofold. No change in the degradation of Ox-LDL was observed.


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  		Table 3. Selective Effect of GM-CSF on Scavenger Receptors in Human Macrophages

Effect of GM-CSF on Scavenger Receptor mRNA Expression in Human Macrophages
Two scavenger receptor isoforms have been described that have similar, if not identical, binding characteristics.37 38 To determine which isoform of scavenger receptor is affected by GM-CSF treatment of human macrophages, mRNA expression of both subtypes was analyzed by Northern blotting. Little or no scavenger receptor mRNA could be detected in lymphocytes (Fig 5Down). Freshly isolated monocytes expressed predominantly scavenger receptor type II mRNA. Differentiation of monocytes into macrophages was associated with a marked increase in type I scavenger receptor transcripts. Macrophages differentiated in the presence of GM-CSF demonstrated a clear reduction in both types I and II transcript, suggesting that the reduction in scavenger receptor activity of GM-CSF found in the binding experiments was also mediated at scavenger receptor mRNA levels.



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  		Figure 5. Effect of GM-CSF on scavenger receptor types I and II expression in human macrophages. Total RNA (10 µg) was separated by formaldehyde-agarose electrophoresis, blotted onto nitrocellulose membranes, and hybridized with an anti-sense oligonucleotide corresponding to exon IV, which encodes for the {alpha}-helical coiled-coil domain of the scavenger receptor and is shared by both scavenger receptor isoforms. A and B, Northern blots of (A) scavenger receptor hybridization and (B) human S18 hybridization. C, Bar graph showing relative distribution of types I (solid bar) and II (open bar) scavenger receptor in lymphocytes (1), monocytes (2), control macrophages (3), and GM-CSF–treated macrophages (4).

Cholesterol Loading of Human Macrophages
The effects of Ac-LDL and Ox-LDL on the cholesterol content of macrophages after differentiation in the absence or presence of GM-CSF are shown in Table 4Down. Macrophages had already accumulated cholesteryl esters during differentiation, probably as the result of the uptake and degradation of modified lipoproteins already present in the serum or lipoproteins modified by the macrophages themselves. Differentiation of macrophages in the presence of GM-CSF was accompanied by a reduction of cholesteryl ester accumulation of 45% compared with control macrophages. Free cholesterol increased 29%. An additional incubation of 4 days with Ac-LDL was followed by an increase of 40% of free cholesterol, while the cholesteryl ester content almost doubled compared with an additional incubation of 4 days in the presence of LPDS only. The accumulation of cholesteryl esters triggered by Ac-LDL was significantly smaller in GM-CSF–treated macrophages than in control macrophages. An additional incubation of 4 days with Ox-LDL was also followed by an increase in free cholesterol and cholesteryl esters and was larger than with Ac-LDL, but no differences were found between macrophages differentiated in the absence or presence of GM-CSF. Four days of incubation with 100 µg/mL Ac-LDL or Ox-LDL reduced protein content per well by 7.6% and 43.0%, respectively.


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  		Table 4. Effect of GM-CSF on the Cholesterol Content of Macrophages


*    Discussion
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 up arrowIntroduction
 up arrowMethods
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*Discussion
down arrowReferences
 
Scavenger receptors are homotrimeric membrane glycoproteins that mediate the uptake of various ligands, including chemically modified lipoproteins, polyribonucleotides, polysaccharides, anionic phospholipids, and other molecules (for a recent review, see Reference 39). In vivo, scavenger receptors are predominantly expressed at the surface of tissue macrophages.40 Scavenger receptors can contribute significantly to the accumulation of cholesterol in macrophages. Unlike the LDL receptor, they are not downregulated in response to an increase in cellular cholesterol.6

Since M-CSF and GM-CSF play a prominent role in the regulation of the activity of mature macrophages, they have the potential to modulate scavenger receptor pathways. Several lines of evidence suggest that M-CSF might play a role in macrophage lipid metabolism. M-CSF stimulates the secretion of lipoprotein lipase and apoE by macrophages.41 42 M-CSF also regulates the activities of neutral and acidic cholesteryl ester hydrolases in human monocyte–derived macrophages43 and enhances the uptake and degradation of Ac-LDL by upregulation of the number of receptors.22 In contrast to M-CSF, few data are available concerning the influence of GM-CSF on the interaction and processing of modified lipoproteins by macrophages. The present investigation focused on possible effects of GM-CSF on receptors that recognize Ac-LDL and/or Ox-LDL at the cell surface of human monocyte–derived macrophages.

The central observation presented here is that the addition of GM-CSF to the culture medium during differentiation of monocytes into macrophages led to a downregulation of the scavenger receptor that recognizes both Ac-LDL and Ox-LDL. Direct evidence was obtained from binding experiments with radiolabeled Ac-LDL and Ox-LDL which showed that upon treatment with GM-CSF the maximal number of binding sites was reduced and the affinity of the ligands for the binding sites unchanged. Treatment of macrophages with GM-CSF resulted in a twofold to threefold reduction in the cell association of radiolabeled modified lipoproteins. Furthermore, the cholesterol content of GM-CSF–treated macrophages in the presence or absence of Ac-LDL was less than that in cells not treated with GM-CSF. This is in agreement with a reduced number of functional scavenger receptors present on the cell surface. Competition experiments demonstrated that the binding site that recognized both Ac-LDL and Ox-LDL was selectively affected when monocytes were treated with GM-CSF during differentiation into macrophages. Northern blot analysis revealed that both types I and II scavenger receptor were markedly reduced upon GM-CSF treatment of the macrophages.

In addition to the types I and II scavenger receptor characterized by Kodama et al,44 additional types of scavenger receptor have been proposed. A 95-kD protein is thought to act as a specific receptor for Ox-LDL,45 whereas a recognition site specific for Ac-LDL has been found in murine peritoneal macrophages.46 The competition experiments described here with adherent human monocyte–derived macrophages suggest that Ox-LDL is recognized by only one site, ie, the Ac-LDL/Ox-LDL binding site. No significant specific Ox-LDL binding activity could be detected under our conditions. A binding site specific for Ox-LDL has been demonstrated in mouse peritoneal macrophages,47 mouse J774 macrophages,48 and rat,45 rabbit,49 and human50 Kupffer cells. However, evidence is weak for a receptor specific for Ox-LDL in human monocyte–derived macrophages using ligand binding and degradation assay experiments; Keidar et al51 have shown that only 10% of the total Ox-LDL degradation is not displaceable by Ac-LDL or native LDL.

The degradation pathway of Ox-LDL resembles that of Ac-LDL, but the apoB of Ox-LDL appears to be more resistant to hydrolysis by lysosomal proteinases than the apoB of Ac-LDL.52 53 54 As the rate-limiting factor for the degradation of 125I-Ox-LDL is likely to be related to lysosomal processing, this might explain the similar degradation values for control and GM-CSF–treated macrophages. The fact that cholesterol loading of macrophages with Ox-LDL was not influenced by GM-CSF treatment of macrophages is also consistent with this explanation.

Two scavenger receptor isoforms with similar binding properties47 have been isolated and cloned from bovine, human, and murine sources.32 37 38 55 Differences in levels of expression between types I and II may be functionally significant. For example, the expression of type I scavenger receptor at both the protein and mRNA levels is specifically increased during the differentiation of human monocytes into macrophages.56 This leads to an increased ratio of type I–to–type II scavenger receptor that is maintained during the transformation of macrophages into foam cells, implying that the regulation of type I macrophage scavenger receptor might play a critical role in foam cell generation.

Other cytokines reduce scavenger receptor expression in macrophages. Fong et al34 have shown that degradation of Ac-LDL is inhibited by interferon gamma in mouse peritoneal macrophages. A similar observation has been made in human monocyte–derived macrophages by Geng and Hansson,35 who also demonstrated that the inhibition by interferon gamma was mediated at the mRNA level. In human THP-1 macrophages scavenger receptor activity as well as mRNA levels are suppressed when transforming growth factor–ß1 is present during differentiation.36 Van Lenten and Fogelman57 have shown that the LPS-induced inhibition of scavenger receptor expression in human monocyte–derived macrophages is mediated through tumor necrosis factor–{alpha}. Interestingly, they also reported that addition of GM-CSF enhances an increase in scavenger receptor function and mRNA after the removal of LPS from the medium. Their study focused on early stages of differentiation of macrophages (3 through 5 days) and on LPS-activated macrophages.57 It is possible that the state of activation as well as degree of differentiation of macrophages affect their responsiveness to GM-CSF and other factors. For instance, very mature macrophages lose the ability to respond to LPS.57 In the aforementioned studies, however, no distinction between type I or II scavenger receptor was made.

The present investigation shows that GM-CSF is capable of downregulating both scavenger receptor types I and II. Types I and II scavenger receptor recognize both Ac-LDL and Ox-LDL, which explains the effect of GM-CSF on the binding of Ac-LDL and Ox-LDL. Our study suggests that type II scavenger receptor expression, like type I,56 is also under a regulatory influence. This regulation could be due to an increase in transcription of the scavenger receptor gene and/or a destabilization of mRNA, which will be the subject for future studies.


*    Selected Abbreviations and Acronyms
 
Ac-LDL = acetylated LDL
GM-CSF = granulocyte macrophage colony-stimulating factor
HSA = human serum albumin
LPDS = lipoprotein-deficient serum
LPS = lipopolysaccharide
M-199 = medium 199
M-CSF = macrophage colony-stimulating factor
Ox-LDL = oxidized LDL
PBS = phosphate-buffered saline
PCR = polymerase chain reaction
poly(I) = polyinosinic acid


*    Acknowledgments
 
The authors acknowledge Sabine Turri for her expert technical assistance and Dr Thomas Giller for providing the S18 probe and for his help in establishing the Northern blot analysis.

Received February 10, 1995; accepted September 15, 1995.


*    References
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*References
 

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