Monocyte colony-stimulating factor enhances uptake and degradation of acetylated low density lipoproteins and cholesterol esterification in human monocyte-derived macrophages.

We have investigated effects of monocyte colony-stimulating factor (M-CSF) on the uptake of acetylated low density lipoproteins (acetyl-LDL) and the activity of cholesterol esterification in human monocyte-derived macrophage. The cells were cultured with M-CSF for 10 days and then incubated with acetyl-LDL for 24 h. M-CSF (128 ng/ml) enhanced the uptake and degradation of 10 micrograms/ml of 125I-acetyl LDL 7.5-fold (n = 6) and the effect of M-CSF was dose-dependent at the concentrations of 0.5-32 ng/ml. The binding experiments at 4 degrees C demonstrated that the number of acetyl-LDL receptor was increased by the addition of M-CSF. Supporting this, ligand blotting analysis revealed a significant increase in a receptor protein for acetyl-LDL (240 kDa). Binding of LDL was also enhanced by M-CSF but less significantly than that of acetyl-LDL. Cellular cholesterol esterification in the presence of 10 micrograms/ml acetyl-LDL was enhanced 24.1-fold (n = 13) by 128 ng/ml M-CSF. It was evident that M-CSF enhanced cholesterol esterification to a greater extent than the cellular uptake of acetyl-LDL (24.1- versus 7.5-fold). Cholesterol esterification was also enhanced by the addition of granulocyte-macrophage colony-stimulating factor and interleukin 1. We conclude that M-CSF enhances the uptake of both acetyl-LDL and LDL by increasing their receptor number, and further enhances the process of cholesterol esterification, resulting in a remarkable increase in cholesterol esterification in macrophages. These findings strongly suggest the significant involvement of cytokines such as M-CSF in cholesterol metabolism of macrophages.

We have investigated effects of monocyte colonystimulating factor (M-CSF) on the uptake of acetylated low density lipoproteins (acetyl-LDL) and the activity of cholesterol esterification in human monocytederived macrophage.
The cells were cultured with M-CSF for 10 days and then incubated with acetyl-LDL for 24 h. M-CSF (128 rig/ml) enhanced the uptake and degradation of 10 pg/ml of '261-acetyl LDL 7.6-fold (n = 6) and the effect of M-CSF was dose-dependent at the concentrations of 0.6-32 rig/ml. The binding experiments at 4 "C demonstrated that the number of acetyl-LDL receptor was increased by the addition of M-CSF.
Supporting this, ligand blotting analysis revealed a significant increase in a receptor protein for acetyl-LDL (240 kDa). Binding of LDL was also enhanced by M-CSF but less significantly than that of acetyl-LDL. Cellular cholesterol esterification in the presence of 10 pg/ml acetyl-LDL was enhanced 24.1fold (n = 13) by 128 rig/ml M-CSF. It was evident that M-CSF enhanced cholesterol esterification to a greater extent than the cellular uptake of acetyl-LDL (24.1versza 7.5-fold). Cholesterol esterification was also enhanced by the addition of granulocyte-macrophage colony-stimulating factor and interleukin 1. We conclude that M-CSF enhances the uptake of both acetyl-LDL and LDL by increasing their receptor number, and further enhances the process of cholesterol esterification, resulting in a remarkable increase in cholesterol esterification in macrophages. These findings strongly suggest the significant involvement of cytokines such as M-CSF in cholesterol metabolism of macrophages. 13). Cloning of the M-CSF gene by Wong et al. (14) enabled a large-scale production of this cytokine in extreme purity and facilitated further studies on its precise function in uivo (8) and in uitro (9)(10)(11)(12)(13). We have recently reported that M-CSF causes a significant reduction in plasma concentrations of cholesterol in young patients with hematological disease (15), suggesting that M-CSF modulates plasma cholesterol metabolism.
Macrophages take up a variety of lipoproteins via receptor mediated endocytosis, resulting in cholesterol accumulation within the cells. LDL and p-very low density lipoproteins are taken up through LDL receptors (16,17), and chemically modified LDL and some oxidized LDL are taken up through acetyl-LDL receptors (l&19). However, little is known about the regulation of lipoprotein receptor activities in macrophages. Fogelman et al. (20) reported time-dependent changes in the activity of receptors for LDL and malondialdehydemodified LDL during the culture of monocytes. They also reported that a lymphokine suppressed receptor activities for malondialdehyde-modified LDL (21,22). In addition, nonphysiological agents such as lipopolysaccharides, muramyl dipeptide, poly(I):poly(C), and b-interferon modulate receptor activities of macrophages (23,24) and sterol content in cells regulates LDL receptor activity (20,25). M-CSF regulates macrophage activity and it is well known that macrophage is one of major cells in atherosclerotic plaque. Thus, it is very possible that M-CSF regulates receptor-mediated uptake of lipoprotein-cholesterol in macrophages and further plays a significant role in atherogenesis.
Here, we present data demonstrating that M-CSF enhances the uptake of acetyl-LDL by increasing the numbers of the receptor.
The human monocyte-specific colony-stimulating factor (CSFl, M-CSF)' is an 85-kDa glycoprotein that stimulates the proliferation and differentiation of monocytic progenitor cells (1,2). Besides its actions as a growth factor, M-CSF stimulates a number of function of mature macrophages (3-  Macrophages were incubated with medium containing 10% autologous serum with (0) or without (0) 128 rig/ml M-CSF. At the 9th day, the medium was removed and the cells were washed three times with PBS and preincubated with medium containing 5 mg/ml LPDS with and without M-CSF for 24 h. After washing with PBS three times, each monolayer received 1 ml of medium containing 5 mg/ml LPDS and varying concentrations of either iz51-acetyl-LDL (A) or iZ51-LDL (B) with or without a 50-fold excess of the respective unlabeled lipoproteins. After 24 h at 37 "C, the amounts of degraded '251-labeled lipoproteins were determined. The values are the means of duplicate wells.
with maximal velocity at 5-10 pg of protein/ml (Fig. L4). 1251-LDL was also taken up and degraded by macrophages by a saturable high affinity process with maximal velocity at 20-30 pg of protein/ml (Fig. 1B). A 50-fold excess of unlabeled acetyl-LDL displaced the degradation of "'1-acetyl-LDL by more than 90% both in the presence and in the absence of M-CSF, while the same order of excess of unlabeled LDL displaced the degradation of '251-LDL by only about 50%. In this experiment, M-CSF augmented the V,., 5-fold for specific degradation of '251-acetyl-LDL and 3-fold for iZ51-LDL, and did not change the K,,, of degradation significantly for '251-acetyl-LDL and decreased it for LDL degradation (51-31 pg/ml). We found that the responsiveness to M-CSF was very variable among macrophages obtained from different donors as shown in Tables I and II. In six experiments observing degradation of 10 rg/ml acetyl-LDL, M-CSF enhanced it 7.5fold, and 4.0-fold for LDL (n = 5), compared to no addition of M-CSF (Table II). It was reported that LDL can be modified, in particular oxidized by secretory products of mac- Relative increases in degradation of radioiodinuted lipoproteins and in cholesterol esterification in macrophuges by the treatment with M-CSF Macrophages were grown for 9 days in the presence or absence of M-CSF (128 rig/ml) and preincubated with LPDS instead of autologous serum for 24 h. After washing with PBS, the cells were incubated with 10 pg/ml acetyl-LDL or LDL for 24 h. The details are described under "Experimental Procedures." Each assay was done in triplicate or quadruplicate wells. The values for M-CSF (+) were divided by the respective values for M-CSF (-) to give the values of relative increases. Each value represents mean + S.E. of several experiments.
Binding of '251-Labeled Lipoproteins to Macrophages-We measured the amounts of '251-labeled lipoproteins bound to macrophages at 4 "C to determine the number of lipoprotein receptors on the cell surface. In the absence of M-CSF, the binding of both 1251-acetyl-LDL (Fig. 3A) and "'1-LDL ( Fig.  3B) followed typical saturation kinetics as a function of ligand concentration.
Scatchard analysis of the binding data of 1251acetyl-LDL revealed a single class of binding sites (B,,,; 2.9 ng/pg of DNA) with an apparent binding affinity (KJ of 0.74 pg/ml calculated by linear regression (Fig. 3A, inset) (34). Scatchard curve of the '251-LDL binding appeared to be concave (Fig. 3A, inset). This might be caused by at least three possible mechanisms: multiple binding sites for 1251-LDL, heterogeneity in '251-LDL and negative cooperativity in 1251-LDL binding to the LDL receptors of macrophages.
When the data were analyzed by simple linear regression, the B,,, and Kd of '251-LDL binding were calculated to be 0.64 ng/pg of DNA and 2.5 pg/ml, respectively.
However, the preincubation with M-CSF increased mainly B,., of lz51-acetyl-LDL binding 4-fold (12.5  respectively) was measured in monocyte-derived macrophages after 10 days of the culture. First, M-CSF was removed from the culture medium at various times to determine whether the presence of M-CSF is necessary during the entire incubation spun to attain its full stimulatory effect. As shown in Fig. 4  least more than 2 days were required for the up-regulation of cholesterol esterification induced either by acetyl-LDL or by LDL, since there was no difference in cholesterol ester formation on Day 2 between in the presence of M-CSF and in its absence. In an attempt to see the time response of M-CSF on mature macrophages, M-CSF was added to culture medium of macrophages which had been incubated without M-CSF for 9 days. As shown in Fig. 6 The variability of the response in human monocytes has been well documented (5,9). Both secretion of M-CSF from cells themselves and their capacity to be activated might be responsible for this variation. Even in macrophages from a single donor, the degree of the stimulatory effects of M-CSF varied, when the cell density was changed (data not shown). On average, M-CSF enhanced acetyl-LDL-cholesterol esterification 24.1-fold (n = 13), and LDL-cholesterol esterification 7.6-fold (n = 12), in the presence of 10 pg/ml of the respective lipoproteins, compared to no addition of M-CSF (Table II). The stimulation of both acetyl-LDL-and LDL-cholesterol esterification by M-CSF was dose-dependent at M-CSF concentrations of 0.5-128 ng/ ml (Fig. 8).
The Effects of Cytokines on Cholesterol Esterification-In addition to M-CSF, there are several cytokines which modulate the function and growth of macrophages and are available in highly pure forms (26) The concentrations of the cytokines used for these experiments were considered to be above those required to exert the maximal effects of each cytokine (26). Although the stimulatory effects of M-CSF in this experiment were less significant than those shown above, these values were still within the variation in responsiveness to M-CSF among macrophage donor as shown in Table II Macrophages were cultured in medium containing 10% of autologous serum without M-CSF. The medium was replaced every 3 days until the day before the experiments. On Day 9, medium was replaced by fresh medium containing 10% of autologous serum. After 24 h, the medium was replaced by medium containing 5 mg/ml LPDS with or without M-CSF and incubated for 24 h. M-CSF was included in the medium 24, 32, and 48 h long before the harvest or was not included at all. Thereafter, the medium was removed, and the cells were washed three times with PBS and incubated with 1 ml of medium containing 0.2 mM ["C]oleate/albumin supplemented with either 10 rg/ml acetyl-LDL (0) or 10 rg/ml LDL (0). After 6 h at 37 "C, the amounts of cholesteryl ["Cloleate in the cells were measured. The values represent the means of duplicate wells.
LDL-cholesterol esterification in this experiment were in the 45th and 30th percentiles of the results listed in Table II, respectively.
Cholesteryl Ester Accumulation-LPDS-preincubated cells were incubated with either acetyl-LDL or LDL for 24 h, then cellular FC and CE were measured (Table IV). Without lipoproteins, M-CSF increased FC 3-fold and CE lo-fold on a DNA basis. In the absence of M-CSF, acetyl-LDL increased the cellular contents of both FC and CE, while LDL increased only FC slightly. Treatment with M-CSF enhanced the acetyl-LDL-induced accumulation of CE (&fold). Cholesterol Efflux-It is possible that macrophages excrete cholesterol which has been taken up, although the overall effect of M-CSF on lipid balance was a net increase in cholesterol ester within the cells. After preincubation with 10 pg of protein/ml of acetyl-LDL for 24 h, cells were incubated with LPDS-containing medium without lipoproteins for 8 h. The amounts of cellular CE decreased as a function of time (Table V). Both net and relative decreases in cellular CE were greater in M-CSF-treated cells than in nontreated cells. Within 8 h, almost 50% of the CE previously accumulated within the cells had disappeared.
Ligund Blotting-The receptor proteins were visualized by incubating a nitrocellulose strip with lz51-acetyl-LDL (Fig. 9). Ligand blots revealed that lz51-acetyl-LDL bound to a protein of 240 kDa specifically both in macrophages grown with M-CSF and in those without it. The intensity of the band was markedly increased by M-CSF treatment. Addition of excess unlabeled acetyl-LDL displaced this band of 240 kDa almost completely.
Therefore, this band might represent receptor protein for acetyl-LDL, although the molecular weight was slightly different from those reported previously (36,37). In addition to this 240-kDa protein, the strips incubated with "?-acetyl-LDL showed several proteins which were less prominent than the 240-kDa protein and could not be displaced by excess unlabeled acetyl-LDL: 60-, 140-, and ZOO-kDa proteins. These results from ligand blots are consistent with the cell culture studies.  (13), and tumor necrosis factor (5) but also the expression of some critical membrane proteins such as Fc receptors (6,7). The stimulatory effects of M-CSF on the uptake of acetyl-LDL and LDL by macrophages are in accordance with those previous observations.
On the other hand, no significant effect of M-CSF on the expression of Ia antigen was reported in vitro (6). Here, we found that M-CSF enhances the uptake of acetyl-LDL and less significantly LDL by increasing the number of receptors. After these lipoproteins were taken up and degraded by macrophages, cholesterol was esterified in the cells. Cholesterol esterification was further enhanced by M-CSF independently from its stimulation by the uptake of lipoproteins, because the enhancement of cholesterol esterification by M-CSF was to a greater extent than that of lipoprotein uptake. Thus M-CSF caused increased accumulation of cholesterol ester within the cells, despite the in- Macrophages were incubated with medium containing 10% autologous serum with the increasing concentrations of M-CSF indicated on the abscissa. After 9 days, the medium was removed and the cells were washed three times with PBS and then incubated with medium containing 5 mg/ml LPDS with the same concentrations of M-CSF for 24 h. The cells were washed with PBS three times and incubated with medium containing 5 mg/ml LPDS and 0. creased efflux of cholesterol from the cells.
There are several candidates of scavenger receptor for modified proteins such as acetyl-LDL, oxidized LDL, and maleyl albumin (38-42); however, their ligand specificity is under debate. Recently, two closely related but distinct molecules for the bovine acetyl-LDL receptor have been cloned (43). Since both can bind acetyl-LDL, it is possible that we observed both receptor activities unseparately as acetyl-LDL receptors. According to previous studies, receptor activities for acetyl-LDL and LDL on macrophages are modulated by several factors (20)(21)(22)(23)(24). These factors, however, could not be found in the physiological condition and their significance in lipoprotein metabolism in macrophages remains unknown. In contrast, M-CSF is widely produced by cells such as fibroblasts, lymphocytes, and macrophages and can be detected even in blood plasma and has many physiological functions (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Furthermore, the potency of M-CSF to stimulate lipoprotein receptor activities seems to exceed that of the other factors reported (23,24). Thus, regulation of lipoprotein uptake through the receptors by M-CSF could play an impor-   tant role in the uptake of lipoproteins in uiuo especially in reticuloendothelial systems such as macrophages and endothelial cells, which are major target cells of M-CSF.
Besides M-CSF, other cytokines such as GM-CSF and interleukin 1 were found to have the similar biological effects on macrophages (Table III). Since these substances including M-CSF are known as growth factors, their effect of increasing lipoprotein receptors might be relevant to their property of stimulating cell proliferation and cell growth. Chait et al. (44,45) reported that growth factors like insulin and plateletderived growth factor increase the LDL receptors of fibroblasts and an increase in LDL receptor precedes cell division, indicating that LDL-cholesterol is required for the construction of the cell structure in growing cells. However, it is well known that monocytes hardly proliferate in response to growth factors unlike cells such as lymphocyte and fibroblast. In fact, during 10 days of culture with M-CSF, DNA content in macrophage was increased only slightly. Even if lipoprotein receptors are up-regulated by M-CSF to supply cholesterol for cell growth and proliferation, the increase in cellular total cholesterol caused by incubation of lipoproteins with M-CSF appears to be too excessive to accomplish adequate supplementation of cholesterol for cell growth and proliferation (Table IV). Especially in case of acetyl-LDL, intracellular content of cholesterol ester was too much after the treatment of M-CSF, whereas the increase in cellular cholesterol ester was moderate in the presence of LDL after M-CSF treatment. We suggest that M-CSF does not increase the lipoprotein receptors of macrophages to provide cholesterol only for their growth but does so for the active elimination of cholesterol from the environment and its storage.
M-CSF significantly enhances, so called, scavenger function of macrophage (3). To aug-ument the cellular uptake of lipoproteins, monocytes and macrophages were incubated with M-CSF for at least 2 days and 1 day, respectively (Figs. 5 and 6). Other functions of macrophages were also enhanced by M-CSF after a long incubation (5,6,11,13). Scavenger function such as acetyl-LDL receptor activity is known to be induced along with the maturation and differentiation of cells from monocyte to macrophage, whereas LDL receptor activity is diminished during their differentiation (Fig. 5,Ref. 20). Since M-CSF stimulates the maturation and differentiation of macrophage (reviewed in Ref. 3), the enhanced acetyl-LDL receptor activity in macrophages after M-CSF treatment could be understood as a result of enhanced maturation and differentiation of macrophages. In fact, cell protein contents were increased by M-CSF despite no significant increase in cell DNA content.
M-CSF enhanced acetyl-LDL receptor activity to a greater extent than LDL receptor activity. This preference for acetyl-LDL to LDL might be explained by three possibilities: 1) the effect of M-CSF on acetyl-LDL receptor activity is greater than that on LDL receptor activity; 2) the accumulation of cholesterol ester from the incubation with lipoproteins for as long as 24 h might selectively down-regulate LDL receptors, since the LDL receptor activity of monocyte/macrophages is regulated by cellular cholesterol (25); 3) the regulation of lipoprotein receptors is modified by secretory substances whose release was provoked by M-CSF (46). The latter two possibilities could be ruled out by the experiments carried out at 4 "C because neither receptor down-regulation, secretion by macrophages, nor modification of lipoprotein in the medium occur at 4 "C. M-CSF increased the maximal binding capacity for both ""I-acetyl-LDL and "'I-LDL, by 4-and 2fold at 4 "C, respectively (Fig. 2). These results support our first hypothesis that M-CSF up-regulate acetyl-LDL receptor activity greater than LDL receptor activity. The degree to which M-CSF stimulates the uptake and degradation of either 1251-acetyl-LDL or ""I-LDL at 37 "C was almost equivalent to the degree to which M-CSF stimulates their binding at 4 "C, but the relative increases in B,,, caused by M-CSF at 4 "C were slightly less than those in degradation of both '*'I-LDL and lZ51-acetyl-LDL. In addition to the increases in lipoprotein receptor numbers, some other factors might be involved in the enhancement of the uptake and degradation of "'Ilabeled lipoproteins at 37 "C, such as receptor recycling and process of degradation. As far as '*"I-LDL uptake is concerned, its metabolic pathway might be altered by some modification of the lipoproteins during the culture, e.g. oxidation, from the LDL receptor to the acetyl-LDL receptor which has greater activity to take up cholesterol in macrophages (47). The stimulation of cholesterol esterification was much greater than that of the uptake and degradation of '*'I-labeled lipoproteins. The activity of the enzyme-esterifying cholesterol (acyl-CoA:cholesterol acyltransferase) might be directly enhanced by M-CSF, apart from its regulation by free cholesterol pool in which influx of cholesterol is derived from endocytosed lipoproteins (48,49).
Although the overall effect of M-CSF was to stimulate the accumulation of cholesterol ester in the presence of either acetyl-LDL or LDL, M-CSF-treated macrophages released substantial amounts of cholesterol into the medium ( Table  V). The mechanism for this cholesterol efflux remains to be clarified. High density lipoprotein receptor is thought to mediate this process (50), but this remains controversial (51,52). In fact, our observation as well as that of Ho et al. (53) indicated that substantial amounts of cholesterol are released from cell to medium containing only LPDS, without high density lipoproteins. It remains to be determined whether M-CSF stimulates the machinery for cholesterol efflux specifically or whether the amount of cholesterol loaded in cells simply regulates the efflux. The former hypothesis is possible, when this is considered in relation to the recent study indicating that high density lipoprotein receptor-mediated cholesterol efflux in fibroblasts is independently regulated by interferon from its effects on cell growth and cell cholesterol contents (54).
Systemic administration of M-CSF as well as GM-CSF has been recently reported to reduce plasma concentrations of LDL cholesterol (15,55). The reduction in plasma LDLcholesterol might be caused by the stimulation of LDL receptor of reticuloendothelial cells such as macrophages by M-CSF, as observed in the present study. It is possible that M-CSF modulates the atherogenesis.
Further studies are necessary to determine whether M-CSF progress or regress atherosclerosis, because roles of scavenger function of foam cells are not known exactly.