Macrophages possess a number of surface receptors that are capable of mediating the internalization of lipoproteins. The low-density lipoprotein (LDL) receptor of human monocyte macrophages recognizes apolipoprotein B-100 and apolipoprotein E and is rapidly regulated in response to changes in intracellular cholesterol levels. In contrast, in J774 macrophages LDL receptor regulation is defective and LDL can cause massive cholesterol accumulation. The ß migrating very low density lipoprotein (ß-VLDL) receptor is poorly regulated by cellular cholesterol concentrations, readily recognizes apolipoprotein E, poorly recognizes apolipoprotein B-100, and is immunologically related to the LDL receptor. The scavenger receptor (acetyl-LDL receptor) appears to have a molecular weight of 250 000 and is not regulated by cellular cholesterol levels. This receptor recognizes LDL that has been chemically or biologically altered. LDL complexes can also enter macrophages and cause cholesterol accumulation. Examples of such complexes are LDL-dextran sulphate complexes, LDL-proteoglycan aggregates, LDL-mast cell granule complexes, LDL-heparin-fibronectin-denatured collagen complexes, and LDL-antibody complexes. The entry of lipoprotein into macrophages by a pathway that is poorly regulated or is not regulated by cellular cholesterol concentrations appears to be a prerequisite for the formation of arterial foam cells.

Atherosclerosis is a disease of arteries, which is both episodic and focal in nature. The earliest morphological event in the development of the atherosclerotic lesion is the diapedesis of blood monocytes into the subendothelial space. Within days to weeks these monocytes convert into lipid-laden macrophages. As a result of the foamy appearance of these cells in histological sections they are often referred to as foam cells. The predominant lipids in these lesions are cholesteryl esters. The accumulation of cholesteryl esters within the macrophage probably results from the receptor-mediated endocytosis of plasma-derived lipoproteins, some of which may have been modified in the artery wall. Several receptors on macrophages capable of mediating these events have been the subject of extensive research in recent years.

On the basis of studies of mouse peritoneal macrophages Brown and Goldstein initially concluded that macrophages lacked LDL receptors (Goldstein et al. 1979). However, several laboratories (Fogelman et al. 1980; Shechter et al. 1981; Traber & Kayden, 1980; Soutar & Knight, 1982) have since demonstrated that human monocyte-macrophages express an active LDL receptor pathway. Despite the presence of normal LDL receptors on these cells exposure to high levels of LDL in vitro did not lead to cholesteryl ester accumulation of the type seen in atherosclerotic lesions (Shechter et al. 1981). Since the LDL receptor pathway is highly regulated to preserve cellular cholesterol homeostasis (Brown & Goldstein, 1986) it was not surprising that normal LDL did not cause cholesteryl ester accumulation in cultured macrophages (Shechter et al. 1981). In an apparent paradox, it has been observed that macrophages in the artery walls of patients that lacked LDL receptors were filled with cholesteryl esters (Buja et al. 1979). Additionally, after injection of radiolabelled LDL into mutant rabbits (Watanabe hereditable hyperlipidemic, WHHL) that are genetically deficient in LDL receptors, the radiolabel rapidly appeared in macrophages in the subendothelial space (Steinberg et al. 1985). These observations suggest that alternative mechanisms account for the accumulation of lipoprotein-derived cholesteryl esters in foam cells.

It has been demonstrated that LDL recovered from human artery walls (Hoff et al. 1979) or from inflammatory fluid in rabbits (Raymond et al. 1985) is more electronegative than circulating LDL. These findings suggest that plasma LDL may be modified in the artery wall. LDL is but one source from which macrophage cholesteryl esters might be derived. Whereas LDL is rich in apolipoprotein B-100, after cholesterol feeding most animals accumulate a cholesteryl-ester-rich particle in their plasma, which is rich in apolipoprotein E. These E-rich lipoproteins float in the ultracentrifuge in the very low density lipoprotein (VLDL) fraction. These particles contain apolipoprotein B as well as apolipoprotein E and migrate in an electrophoretic field with characteristics intermediate between LDL and VLDL. Hence these particles are called beta migrating VLDL or β-VLDL. These particles also accumulate in the plasma of patients with familial type III hyperlipoproteinemia (Hui et al. 1984).

While LDL was recognized poorly by mouse peritoneal macrophages Goldstein eí al. (1980) found that ß-VLDL was taken up by a high-affinity receptor on these cells. This receptor was poorly down-regulated compared to the LDL receptor on fibroblasts and consequently it was named the ß-VLDL receptor. In other experiments these investigators found that β-VLDL also stimulated cholesteryl ester synthesis in human monocyte-macrophages while LDL did not (Mahley et al. 1980). In 1982 Gianturco et al. showed that ß-VLDL, but not LDL, effectively competed for the uptake of hypertriglyceridemic VLDL (VLDL taken from hypertriglyceridemic subjects) by mouse peritoneal macrophages: these results suggested that the hypertriglyceridemic VLDL was also recognized by the ß-VLDL receptor. In the following year Van Lenten et al. (1983) demonstrated that postprandial lipoproteins from normal fat-fed volunteers, but not their fasting lipoproteins, effectively competed for the uptake of ß-LVLDL by human monocyte-macrophages. Van Lenten et al. (1983) also showed that monocytes from a child with 5 % of the normal number of LDL receptors poorly recognized LDL, but recognized ß-VLDL normally, suggesting that the LDL and ß-VLDL receptors were genetically distinct. In 1984 Baker et al. reported that the uptake of LDL by aortic endothelial cells was dependent upon cellular density, but that the uptake of β-VLDL was not. Baker et al. (1984) also demonstrated that aortic endothelial cells from the mutant WHHL rabbits, which are deficient in LDL receptors, took up LDL poorly but internalized β-VLDL normally, providing what was thought to be further evidence that the LDL and ß-VLDL receptors are genetically distinct.

Reasoning that if excess cholesterol enters into macrophages in the artery wall by receptor-mediated processes, Van Lenten et al. (1985a) studied the receptormediated uptake of lipoproteins in sterol-loaded human monocyte-macrophages. These investigators found that under these conditions of cholesterol loading there was no evidence for receptor-mediated uptake of chylomicrons (the large triglyceride-rich lipoproteins formed in the intestine after a meal) or LDL. These data were consistent with the Brown and Goldstein model, which predicts down-regulation of LDL receptors with cellular cholesterol loading. However, the sterol-loaded human monocyte-macrophages internalized ß-VLDL and chylomicron remnants (postprandial particles produced by the metabolism of circulating chylomicrons that are acted upon by lipoprotein lipase: this enzyme removes triglyceride and results in the production of remnants, which are relatively enriched in cholesteryl esters and apolipoprotein E) by a lower affinity, but specific and saturable, process that was dependent on apolipoprotein E (Van Lenten etal. 1985a). Van Lenten et al. (1985a) also demonstrated that macrophages from WHHL rabbits recognized LDL poorly, but recognized jß-VLDL normally, providing what was thought to be even further evidence that the LDL receptor and the β-VLDL receptor were genetically distinct. Subsequently, Ishii et al. (1986) also reported that ß-VLDL, but not LDL, stimulated cholesteryl ester synthesis in WHHL macrophages.

All of the evidence up to this point indicated that the LDL receptor and the ß-VLDL receptor were distinct. However, in, 1986 Koo et al. found that ligand blots of Triton X-100 extracts of mouse peritoneal macrophages identified a single protein when probed with 125I-ß-VLDL. The protein cross-reacted with antibodies against the bovine LDL receptor. The molecular weight of this receptor was approximately 5000 less than that of the human fibroblast LDL receptor. These investigators also demonstrated that pre-incubation of the mouse macrophages with LDL receptor antibody inhibited the binding of both ß-VLDL and LDL to the cells and also inhibited lipoprotein-induced enhancement of cholesteryl ester synthesis. Moreover, the binding of 125I-ß-VLDL to the mouse macrophages correlated directly with the amount of protein recognized on immunoblots with antibody to the bovine LDL receptor. They concluded that the ß-LVLDL receptor on mouse peritoneal macrophages was an unusual LDL receptor that binds LDL poorly, but binds apolipoprotein E-containing lipoproteins with very high affinity, and this receptor is resistant to down-regulation by intracellular cholesterol (Koo et al. 1986).

The following year Ellsworth et al. (1987) reported that the uptake of 125I-ß-VLDL and 125I-chylomicron remnants in murine macrophage cell lines was competitively inhibited by specific polyclonal antiserum directed against the LDL receptor of rat liver. The anti-LDL receptor immunoglobulin G (IgG), 125I-ß-VLDL, and 1251-chylomicron remnants bound to two protein components, of apparent molecular weights of 125 000 and 111 000, on nitrocellulose blots of detergent-solubilized macrophage membranes. Between 70% and 90% of 125I-lipoprotein binding was confined to the 125K (K = 103Mr) peptide. Binding of 125I-ß-VLDL and 1251-chylomicron remnants to these proteins was competitively inhibited by anti-LDL receptor antibodies. The authors concluded that ß-VLDL and chylomicron remnants were recognized by murine macrophages by an LDL receptor that is immunologically related to the LDL receptor of rat liver (Ellsworth et al. 1987).

The experiments in Fig. 1 show that when human monocyte-macrophages were cholesterol-loaded there was no detectable specific uptake of LDL. Under the same conditions of cholesterol loading, β-VLDL uptake was decreased by approximately 50 %, but even under these conditions ß-VLDL uptake exceeded LDL uptake in the absence of cholesterol loading. However, the uptake of both ß-VLDL and LDL was inhibited under both control and cholesterol loading conditions when anti-bovine LDL receptor IgG was present. Pre-immune IgG had no effect. These experiments indicate that the ß-VLDL receptor of human monocyte-macrophages is immunologically related to the LDL receptor.

Fig. 1.

The effect of anti-bovine adrenal LDL receptor IgG on the degradation of 125I-rabbit β-VLDL and 125I-human LDL in human monocyte-macrophages pre-incubated in the presence or absence of sterols in the medium. Normal human monocytes were cultured in 30% autologous serum as previously described (Van Lenten et al. 1985a). After 4 days the medium was removed and the cells were transferred to 0·l % human serum albumin in 0·5 ml of serum-free medium supplemented with 1 μgml-1 25-hydroxycholesterol and 16 μgml-1 cholesterol in ethanol (bottom panel, sterol-loaded) or with ethanol alone (top panel, ethanol control) as described previously (Van Lenten et al. 1985a). On day 7, the medium was removed and the cells were washed three times. The cells were then incubated with 0·5ml of medium containing 4 μgml-1 125I-rabbit ß-VLDL (cross hatched bars) or 4μg ml-1 125I-human LDL (stippled bars) in the presence or absence of a 100-fold excess of non-radioactive lipoprotein and in the absence (None) or presence of 200 μgml-1 preimmune IgG (APreimmune Serum) or in the presence of 200 μgml-1 anti-bovine adrenal LDL receptor IgG (+αB, E Receptor). After 5h at 37 °C the medium was removed and the 1251-labelled acid-soluble content determined as previously described (Van Lenten et al. 1985a). The values shown are the mean ± 1 S.D. of quadruplicate wells.

Fig. 1.

The effect of anti-bovine adrenal LDL receptor IgG on the degradation of 125I-rabbit β-VLDL and 125I-human LDL in human monocyte-macrophages pre-incubated in the presence or absence of sterols in the medium. Normal human monocytes were cultured in 30% autologous serum as previously described (Van Lenten et al. 1985a). After 4 days the medium was removed and the cells were transferred to 0·l % human serum albumin in 0·5 ml of serum-free medium supplemented with 1 μgml-1 25-hydroxycholesterol and 16 μgml-1 cholesterol in ethanol (bottom panel, sterol-loaded) or with ethanol alone (top panel, ethanol control) as described previously (Van Lenten et al. 1985a). On day 7, the medium was removed and the cells were washed three times. The cells were then incubated with 0·5ml of medium containing 4 μgml-1 125I-rabbit ß-VLDL (cross hatched bars) or 4μg ml-1 125I-human LDL (stippled bars) in the presence or absence of a 100-fold excess of non-radioactive lipoprotein and in the absence (None) or presence of 200 μgml-1 preimmune IgG (APreimmune Serum) or in the presence of 200 μgml-1 anti-bovine adrenal LDL receptor IgG (+αB, E Receptor). After 5h at 37 °C the medium was removed and the 1251-labelled acid-soluble content determined as previously described (Van Lenten et al. 1985a). The values shown are the mean ± 1 S.D. of quadruplicate wells.

All of the above studies are consistent with the recognition of ß-LVLDL, hypertriglyceridemic VLDL, postprandial VLDL, and chylomicron remnants by a receptor on macrophages that poorly recognizes apolipoprotein B-100 (the apolipoprotein of LDL), but which recognizes apolipoprotein E with high affinity.

Hobbs et al. (1986) recently reported a mutation in a family with familial hypercholesterolemia in which the deletion of an exon encoding for a cysteine-rich repeat in the LDL receptor resulted in a receptor that failed to recognize apolipoprotein B-100, but which recognized apolipoprotein E with high affinity. These authors proposed that the ability to bind the single copy of apolipoprotein B-100 in LDL may require a precise arrangement of multiple binding sites, an arrangement that is not possible when one of the sites is deleted. They hypothesized that the binding of ß-VLDL, on the other hand, may be more flexible since each ß-VLDL particle contains multiple copies of apolipoprotein E, which might be able to rearrange themselves on the surface of the particles so as to conform to an altered receptor. Another possibility was also mentioned, that the binding of LDL might require some type of oligomerization of the LDL receptor, whereas binding of β-VLDL might not require such complex formation. Deletion of a cysteine-rich repeat might prevent oligomerization and thus prevent LDL binding without affecting ß-VLDL binding (Hobbs et al. 1986).

Yamamoto et al. (1986) have shown that the defect in the LDL receptor of the WHHL rabbit is a deletion in the cysteine-rich ligand-binding domain of the receptor, which impedes transport of the receptor to the cell surface. A similar defect has been seen in a human (Yamamoto et al. 1986). These defects allow approximately 5 % of the receptors to reach the cell surface. If the deletion of the cysteine-rich region of the WHHL rabbit and some familial hypercholesterolemia homozygotes (e.g. the child in the studies of Van Lenten et al. (1983)) are similar to that described by Hobbs etal. (1986) one might have sufficient receptor to bind the apolipoprotein E in β-VLDL without binding apolipoprotein B-100 (LDL). If this were indeed the case many of the apparent contradictions in the studies reported above would be resolved. These issues await further research. However, regardless of the mechanism by which β-VLDL and chylomicron remnants are internalized by macrophages, these lipoproteins may be important in producing cholesteryl ester accumulation in arterial macrophages.

In 1976 Goldstein and Brown observed that treatment of LDL with acetic anhydride caused the acetylation of the e-amino groups of the lysine residues in LDL. This chemical modification rendered the LDL more electronegative and prevented the lipoprotein from being recognized by the LDL receptor (Basu et al. 1976). Consequently, LDL treated with acetic anhydride (acetyl-LDL) was poorly taken up by fibroblasts. However, in, 1979 Goldstein et al. found that acetyl-LDL was avidly taken up by mouse peritoneal macrophages, while native LDL was poorly recognized and internalized. As a consequence of the marked internalization and degradation of the acetyl-LDL, the mouse peritoneal macrophages became massively enriched in cholesteryl esters (Goldstein et al. 1979). Unfortunately, extensive studies failed to identify an in vivo process that could produce such acetylation. In, 1980 Fogelman et al. found that malondialdehyde (which is produced in vivo by the metabolism of arachidonic acid and by lipid peroxidation) reacted with LDL in vitro to produce a more electronegative particle. This malondialdehyde-treated LDL (MDA-LDL) was not recognized by the LDL receptor of human monocytemacrophages and appeared to be recognized by a different receptor on the human monocyte-macrophages (Fogelman et al. 1980). This receptor was shown to be the same receptor that recognized acetyl-LDL and it was also found to produce massive cholesteryl ester accumulation in human monocyte-macrophages (Shechter et al. 1981). This receptor became known as the scavenger receptor as it seemed to recognize a number of negatively charged proteins and compounds (Brown et al. 1980).

The activity of this scavenger receptor was found to be independent of cellular cholesterol concentrations (Fogelman et al. 1981). Scavenger receptor activity was low in freshly isolated human monocytes but increased 10-fold as the monocytes converted into macrophages in culture (Fogelman et al. 1981). Experiments indicated that the scavenger receptor and the LDL receptor were on the same cell (Fogelman et al. 1981). However, LDL receptor activity declined with increasing cell density while scavenger receptor activity increased with increasing cell density (Fogelman et al. 1981; Mazzone & Chait, 1982). Lymphokines prevented the expression of the scavenger receptor as did endotoxin (Fogelman et al. 1982, 1983; Van Lenten et al. 1985b, 1986). Diphosphoryl Lipid A was found to be the active principal in preventing the expression of this receptor on human monocytemacrophages (Van Lenten et al. 1985b). The entry of endotoxin into human monocytes and the subsequent suppression of scavenger receptor activity appeared to depend on formation of an endotoxin-LDL complex followed by the receptor-mediated endocytosis of this complex via the LDL receptor pathway (Van Lenten et al. 1986). The only agent thus far identified that increases scavenger receptor activity in cultured human monocyte-macrophages is dexamethasone (Hirsch & Mazzone, 1986).

The scavenger receptor has only been found on monocyte-macrophages, Kupffer cells and on endothelial cells (Baker et al. 1984; Stein & Stein, 1980; Dresel et al. 1985; Pitas et al. 1985; Netland et al. 1985; Nagelkerke et al. 1983; Horiuchi et al. 1985a), particularly hepatic sinusoidal cells (Pitas et al. 1985; Netland et al. 1985; Nagelkerke et al. 1983; Horiuchi et al. 1985a).

Goldstein and Brown had suggested that scavenger receptor recognition resulted primarily from protein modification by reagents that produced a more electronegative protein (Goldstein et al. 1980; Brown et al. 1980). Such modifications included acetylation with acetic anhydride, aceto-acetylation, Schiff’s base formation with malondialdehyde, maleylation with maleic anhydride or succinylation with succinic anhydride. Subsequently, Haberland et al. (1982, 1984) and Haberland & Fogelman (1985) demonstrated that negative charge was involved in receptor recognition, but that negative charge alone was not sufficient for receptor recognition. Indeed, the reagents employed appeared to alter the protein so that clusters of negative charges appeared due to conformational shifts in the protein. Moreover, at least in the case of one ligand, maleyl-albumin, removal of the reagent (i.e. de-maleylation) left a conformationally altered protein, which was still recognized by the scavenger receptor even though the de-maleylated protein had lost its electronegativity (Haberland & Fogelman, 1985). This suggested that the primary sequence of albumin could be induced, by a conformational change, to expose clusters of negative charge that were recognized by the scavenger receptor, even though the overall charge of the protein was not different from native albumin, which is not recognized by the scavenger receptor (Haberland & Fogelman, 1985). In support of this hypothesis was the finding that changing the conformation of the de-maleylated protein by exposure to guanidine HC1 resulted in a loss of receptor recognition (Haberland & Fogelman, 1985).

Haberland et al. also demonstrated that some reagents that modify lysine residues are more efficient than others in producing molecules that are recognized by the scavenger receptor. The modification of only 16% of the lysine residues in LDL by malondialdehyde produces scavenger receptor recognition (Haberland et al. 1982) while approximately two thirds of the lysine residues need to be modified by acetylation with acetic anhydride in order to achieve a similar degree of scavenger receptor recognition (Haberland et al. 1984).

Oxidation of LDL to a form recognized by the scavenger receptor via a metal-ion-dependent process and/or by cell-dependent processes have also been demonstrated in several laboratories (Morel et al. 1984; Henrickson et al. 1981; Parthasarathy et al. 1985; Heinecke et al. 1986). Cultured endothelial cells (Morel et al. 1984; Henrickson et al. 1981; Parthasarathy et al. 1985), and arterial smooth muscle cells (Heinecke et al. 1986), have been shown to be capable of oxidizing LDL to a form recognized by the scavenger receptor. Steinbrecher (1987) has recently shown that products of LDL lipid oxidation are capable of modifying the lysine residues of LDL and hence may induce a conformational change that leads to recognition of the modified lipoprotein by the scavenger receptor. Products of lipid oxidation that might mediate such lysine modification include 4-hydroxynonenal (Jurgens et al. 1985) and malondialdehyde (Fogelman et al. 1980). To date, only malondialdehyde has been shown to be present in vivo where it co-localized with apolipoprotein B in the atherosclerotic lesions of the WHHL rabbit (Haberland et al. 1988). Kita et al. (1987) recently demonstrated that probucol, an antioxidant, prevented the progression of atherosclerosis in WHHL rabbits. They hypothesized that this agent limited oxidative LDL modification and hence prevented foam cell formation (Kita et al. 1987).

The scavenger receptor has neither been purified to homogeneity nor has its normal function been established. Phillips et al. (1985) have demonstrated that thrombin-activated platelets secrete products that prevent the uptake and hydrolysis of modified LDL by macrophages. However, the natural ligand(s) of the scavenger receptor is unknown and probably will remain unknown until the receptor is purified. Partial purifications of the scavenger receptor have indicated that this protein has a molecular weight of approximately 250 000 (Via et al. 1985; Kodama & Krieger, 1986; Dresel et al. 1987). Figs 2, 3 demonstrate that binding activity can be recovered from human monocyte-macrophages in an HPLC fraction, which contains a 250K protein that is seen on a ligand blot in the absence but not in the presence of a competitive inhibitor of the scavenger receptor. While this 250K protein may well be the scavenger receptor, it should be noted that at least two other macrophage proteins have been identified that can also bind ligands of the scavenger receptor (Horiuchi et al. 1985a,b,c;Vlassara et al. 1986). These receptors have been presumed to be different from the scavenger receptor based on cross-competition experiments. However, both the receptor for formaldehyde-treated serum albumin (Horiuchi et al. 1985a,b,c) and the receptor for glucose-modified proteins (Vlassara et al. 1986) bind acetyl-LDL. Thus, it is possible that these receptors in vivo could also mediate the uptake of scavenger receptor ligands.

Fig. 2.

Partial purification of the human monocyte-macrophage scavenger receptor. Human monocyte-macrophages were cultured in 30 % autologous serum as described previously (Van Lenten et al. 1985a). After 7 days the medium was removed and the cells were washed three times with phosphate buffered saline (PBS) and then lysed with 15 ml of buffer containing 1 % NP-40, 0·5 % sodium deoxycholate, 150 mM-NaCl, 50mM-Tris pH8·3, 10mM-EDTA, and protease inhibitors (30 μgml-1 aprotinin, 20 μgml-1 pepsta-tin, and 0·5 mM-phenylmethylsulphonylfluoride). All subsequent buffers contained these protease inhibitors. The lysate was applied to an ion exchange (PEI) column and eluted essentially as described by Via et al. (1985). The eluate was then subjected to gel permeation chromatography on a TSK 400 HPLC column essentially as described by Dresel et al. (1987). The phosphatidylcholine liposome filter assay to measure 125I-maleyl-albumin binding to HPLC fractions was performed essentially as described by Via et al. (1985). Absorbance is indicated on the left ordinate and the binding of 125I-maleylalbumin is shown on the right ordinate (open circles).

Fig. 2.

Partial purification of the human monocyte-macrophage scavenger receptor. Human monocyte-macrophages were cultured in 30 % autologous serum as described previously (Van Lenten et al. 1985a). After 7 days the medium was removed and the cells were washed three times with phosphate buffered saline (PBS) and then lysed with 15 ml of buffer containing 1 % NP-40, 0·5 % sodium deoxycholate, 150 mM-NaCl, 50mM-Tris pH8·3, 10mM-EDTA, and protease inhibitors (30 μgml-1 aprotinin, 20 μgml-1 pepsta-tin, and 0·5 mM-phenylmethylsulphonylfluoride). All subsequent buffers contained these protease inhibitors. The lysate was applied to an ion exchange (PEI) column and eluted essentially as described by Via et al. (1985). The eluate was then subjected to gel permeation chromatography on a TSK 400 HPLC column essentially as described by Dresel et al. (1987). The phosphatidylcholine liposome filter assay to measure 125I-maleyl-albumin binding to HPLC fractions was performed essentially as described by Via et al. (1985). Absorbance is indicated on the left ordinate and the binding of 125I-maleylalbumin is shown on the right ordinate (open circles).

Fig. 3.

Binding of malondialdehyde-altered LDL (MDA-LDL) by partially purified human monocyte-macrophage scavenger receptor. Proteins from fraction 7 of the experiment described in Fig. 2 were separated on a 6% polyacrylamide gel under nondenaturing conditions essentially as described by Via et al. (1985). They were then electrophoretically transferred to nitrocellulose. Non-specific protein binding sites on the nitrocellulose strips were blocked with non-fat dry milk. The nitrocellulose strips were incubated with 100 μgml-1 MDA-LDL in the absence (-) or presence (+) of 500 μgml-1 polystyrene sulphonate. Bound MDA-LDL was detected by exposing the strips to an affinity-purified rabbit antibody to MDA-LDL. This was followed by exposing the strips to biotin-labelled goat anti-rabbit IgG. After washing, the strips were exposed to avidin complexed with biotinvlated alkaline phosphatase. Finally, the strips were washed and developed for colour with the alkaline phosphatase reagents 5-bromo-4-chloro-3-indolylphosphate ρ toluidine salt and nitroblue tetrazolium chloride.

Fig. 3.

Binding of malondialdehyde-altered LDL (MDA-LDL) by partially purified human monocyte-macrophage scavenger receptor. Proteins from fraction 7 of the experiment described in Fig. 2 were separated on a 6% polyacrylamide gel under nondenaturing conditions essentially as described by Via et al. (1985). They were then electrophoretically transferred to nitrocellulose. Non-specific protein binding sites on the nitrocellulose strips were blocked with non-fat dry milk. The nitrocellulose strips were incubated with 100 μgml-1 MDA-LDL in the absence (-) or presence (+) of 500 μgml-1 polystyrene sulphonate. Bound MDA-LDL was detected by exposing the strips to an affinity-purified rabbit antibody to MDA-LDL. This was followed by exposing the strips to biotin-labelled goat anti-rabbit IgG. After washing, the strips were exposed to avidin complexed with biotinvlated alkaline phosphatase. Finally, the strips were washed and developed for colour with the alkaline phosphatase reagents 5-bromo-4-chloro-3-indolylphosphate ρ toluidine salt and nitroblue tetrazolium chloride.

Maleyl-albumin was one of the original ligands of the scavenger receptor described by Goldstein et al. (1979). Recently, Haberland et al. (1986a,b) demonstrated in freshly isolated human monocytes that this ligand was also recognized by a different receptor, which mediates monocyte chemotaxis.

Yet another mechanism for the unregulated uptake of cholesteryl-ester-rich particles by macrophages was described by Basu et al. in 1979, the dextran sulphate pathway. They found that the uptake and degradation of 125I-LDL by mouse peritoneal macrophages was increased markedly by the addition of high-molecular-weight dextran sulphate. The dextran sulphate and 125I-LDL formed a complex, which was taken up and degraded with saturation kinetics suggesting a specific cell-surface high-affinity binding site. Competition studies indicated that this was neither the LDL receptor nor the scavenger receptor. These experiments indicated that macrophages have the capacity to ingest large amounts of LDL in association with high-molecular-weight sulphated polysaccharides and that this ingestion can lead to cholesteryl ester deposition in these cells.

Vijayagopal et al. (1985) later demonstrated that proteoglycan aggregates extracted from bovine aorta, when complexed to LDL in vitro in the presence of Ca2+, were taken up and degraded by mouse peritoneal macrophages by a high-affinity saturable process. The uptake of these complexes was inhibited by unlabelled acetyl-LDL, but not LDL or polyinosinic acid or fucoidin, leaving open the question of which receptor mediated the uptake of these complexes. Exposure of the mouse peritoneal macrophages to the proteoglycan aggregate-LDL complex produced increased cholesteryl ester synthesis and massive cholesteryl ester accumulation.

Kokkonen & Kovanen (1987) recently demonstrated that stimulation of mast cells can lead to the formation of LDL-granule complexes which can be phagocytosed by macrophages and cause cholesterol accumulation.

Falcone et al. (1984) prepared insoluble complexes of LDL, heparin, fibronectin and dentured collagen (gelatin) and found that this complex was taken up by mouse peritoneal macrophages at a slower rate than LDL or acetyl-LDL and was degraded less effectively, resulting in an accumulation of undegraded cholesteryl esters within the cell. The cell cytoplasm was filled with phagosomes containing material similar in appearance to the LDL-matrix complexes rather than the lipid droplets characteristic of foam cells.

Another pathway that might mediate cholesteryl ester accumulation in macrophages is the Fc receptor pathway. Witztumct al. (1984) demonstrated the presence of auto-antibodies to glycosylated proteins in the plasma of patients with diabetes. Glycosylated LDL-antibody complexes could be cleared by the Fc receptor. Since the Fc receptor is not regulated by cellular cholesterol levels this could lead to macrophage cholesteryl ester accumulation (Brown & Goldstein, 1983; Klimov et al. 1985).

The LDL receptor of the J774 cell appears to be poorly regulated as shown by Tabas et al. (1985) who found that unmodified LDL caused cholesteryl ester accumulation in J774 macrophages. They reported that LDL was internalized by the LDL receptor but in comparison with fibroblasts, the LDL receptor and HMG-CoA reductase activity in J774 cells were relatively resistant to down regulation by LDL or 25-hydroxycholesterol. Consequently the cells continued to take up LDL via the receptor and to accumulate cholesteryl esters. In addition, J774 cells appeared to accumulate cholesteryl esters from LDL internalized by non-specific processes. Subsequently, Tabas et al. (1986) found that inhibition of the enzyme acyl coenzyme A:cholesterol acyl transferase (ACAT) in J774 macrophages enhanced downregulation of the LDL receptor and HMG-CoA reductase in these cells, and consequently prevented LDL-induced cholesterol accumulation. In contrast to the findings with J774 macrophages, down-regulation of the human fibroblast LDL receptor was not enhanced by their ACAT inhibitor. They concluded that in J774 macrophages, but not in fibroblasts, ACAT competes for a regulatory pool of intracellular cholesterol, and results in diminished receptor and reductase downregulation, which in turn results in cellular LDL-cholesterol accumulation, and foam cell formation (Tabas et al. 1986).

Wolfbauer et al. (1986) and Minor et al. (1986) have demonstrated that nonreceptor-mediated uptake may also lead to massive cholesteryl ester accumulation. They exposed smooth muscle cells to cholesteryl-ester-rich lipid droplets extracted from macrophages. After repeated failure to transfer the cholesteryl esters in the medium to the smooth muscle cells, they cultured the smooth muscle cells on coverslips and then inverted these on the medium containing the lipid droplets. The result was massive cholesteryl ester accumulation. Apparently the buoyant lipid droplets floated to the surface under standard culture conditions and, consequently, they did not come in contact with the smooth muscle cells. In the artery wall the lipid droplets released from dying macrophages would probably be trapped in the collagen network and could come into contact with the smooth muscle cells. Such a scenario would explain the observed early appearance of cholesterol-laden macrophage cells and the later appearance of cholesterol-loaded smooth muscle cells.

Both receptor-mediated and non-receptor-mediated processes can deliver cholesteryl-ester-rich molecules into macrophages. A better understanding of these processes will advance our understanding of macrophage biology and may lead to more rationale therapies for atherosclerosis.

We thank Fara Elahi and Ken Ho for excellent technical assistance in preparing human monocytes and Susan C. Murphy for the preparation of this manuscript. We thank Dr Tom Innerarity of the Gladstone Foundation (San Francisco, CA) for the gift of the anti-bovine adrenal LDL receptor IgG. This work was supported in part by United States Public Health Service Grants HL 30568, IT32 HL 07412, and RR 865, the Laubisch Fund, and the M. K. Grey Fund.

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