(Received for publication, November 5, 1996, and in revised form, February 3, 1997)
From the Department of Medicine, The University of
British Columbia, Vancouver, British Columbia, V5Z 4E3 Canada and
§ Department of Molecular Biology and Medicine, Research
Center for Advanced Science and Technology, University of Tokyo,
4-6-1 Komaba, Meguro, Tokyo 153, Japan
Oxidation of low density lipoproteins (LDL) has
been implicated as a causal factor in the pathogenesis of
atherosclerosis. Oxidized LDL has been found to exhibit numerous
potentially atherogenic properties in vitro, including
receptor-mediated uptake by macrophages. Oxidized LDL is a ligand for
the class A scavenger receptor type I/II (SR-AI/II), but
cross-competition studies with cultured macrophages suggested that
there is an additional receptor(s) that is specific for oxidized LDL
and that does not interact with acetyl LDL or other chemically modified
LDL. A number of macrophage membrane proteins, including CD36,
FcRII-B2, scavenger receptor BI, and macrosialin/CD68, have been
found to bind to oxidized LDL in vitro and have been
proposed as candidate oxidized LDL receptors. However, because of
overlapping ligand specificity with the SR-AI/II, it has been difficult
to evaluate the relative importance of these proteins in the uptake of
oxidized LDL by macrophages. In the present report, we have studied the
uptake and degradation of oxidized LDL by macrophages from mice in
which the SR-AI/II gene had been disrupted. The uptake of acetyl LDL
was reduced by more than 80% in macrophages from scavenger receptor
knockout mice, confirming that most of the uptake of acetyl LDL by
macrophages can be attributed to this receptor. In contrast, the uptake
of extensively oxidized LDL was reduced by only 30% and showed high affinity, saturable uptake with apparent Km of
about 5 µg/ml, similar to that of the SR-AI/II. This indicates that about 70% of the uptake of oxidized LDL in macrophages is attributable to an alternate oxidized LDL receptor(s). In contrast to findings reported with CD36, mildly oxidized LDL was internalized much more
slowly than extensively oxidized LDL. Unlabeled oxidized LDL,
polyinosinic acid, phosphatidylserine-rich liposomes, and LDL or bovine
albumin modified by fatty acid oxidation products were effective
competitors for the uptake of radioiodinated oxidized LDL by
macrophages from knockout mice, whereas acetyl LDL and malondialdehyde-modified LDL were relatively poor competitors. This
ligand specificity differs from that of CD36-related (class B)
scavenger receptors but is similar to the reported specificity of
macrosialin/CD68 in ligand blots. However, the rate of uptake of
oxidized LDL by knockout macrophages was not increased by phorbol ester
or in thioglycollate-elicited macrophages, both of which are expected
to increase the amount of macrosialin on the cell surface. In
macrophages from SR-AI/II knockout mice, ligand blots of membrane
proteins with iodinated, oxidized, or acetylated LDL revealed several
bands, with apparent molecular size on SDS-polyacrylamide gel
electrophoresis of 60, 94, 124, and 210 kDa, but none of the bands were
specific for oxidized LDL. These results provide direct evidence that a
receptor other than SR-AI/II is responsible for most of the uptake of
oxidized LDL in murine macrophages, but further studies are needed to
identify the receptor(s) involved.
Oxidized low density lipoprotein (LDL)1 has been shown to have many potentially atherogenic actions and properties (1-12), and there is evidence that at least a mild degree of oxidation of LDL occurs in arterial lesions in vivo (13-17). Furthermore, there is considerable experimental and epidemiological evidence to suggest a causal role for LDL oxidation in the pathogenesis of atherosclerosis (18-33).
One of the properties that distinguishes oxidized LDL from native LDL is the ability to bind to "scavenger" receptors on macrophages (34, 35). It has been postulated that this could lead to unregulated cholesterol accumulation in macrophages, resulting in the formation of foam cells (1, 36). The term scavenger receptor has been applied to a number of different cell-surface proteins that can bind to chemically or biologically modified lipoproteins and/or senescent cells. The first scavenger receptor to be fully characterized is termed the class A type I/type II scavenger receptor, or SR-AI/II (37). Two forms of the receptor are produced from a single gene through alternative splicing of mRNA (38, 39). The SR-AI has six structural domains, including a collagen-like domain (V) that mediates ligand binding (40). The cysteine-rich extracellular domain VI is deleted in the type II receptor, without evident effect on binding of most ligands (41). Various negatively charged substances including polyinosinic acid, maleylated albumin, and acetyl LDL can bind to this receptor, suggesting that ligand binding may be mediated principally by ionic interactions (40, 42). It has been shown that a particular conformation of charges is required for receptor binding, because only polynucleotides that are capable of forming base-quartet-stabilized helices act as ligands (43).
Several groups have reported evidence of a receptor for oxidatively
modified LDL distinct from the SR-AI/II on macrophages (44, 45) or
Kupffer cells (46-48). In several of these studies, the evidence for
multiple receptors was the finding that acetyl LDL was able to compete
for only about 40% of oxidized LDL uptake (44-47). More direct
evidence for a separate receptor for oxidized LDL was the finding that
in rats, intravenously injected oxidized LDL was cleared preferentially
by Kupffer cells, whereas acetyl LDL was cleared by sinusoidal
endothelial cells (46). Endemann and co-workers (49, 50) used a mouse
cDNA library and an expression cloning strategy with fluorescently
labeled oxidized LDL to identify two cell surface proteins that could
mediate the binding and internalization of oxidized LDL but not acetyl
LDL. The first was found to be the murine FcII-B2 receptor, but
blocking antibodies to the Fc
II-B2 receptor failed to inhibit the
uptake of oxidized LDL in mouse peritoneal macrophages; hence, it is
unlikely that this receptor contributes significantly to oxidized LDL
uptake in these cells (49). The second protein was shown to be the
murine homologue of CD36 (50). It was found that only a mild degree of
oxidation of LDL was required for maximal binding to the murine
homologue of CD36, whereas extensive oxidation of LDL is required for
receptor-mediated uptake and degradation in mouse peritoneal
macrophages (35). This is indirect evidence that CD36 does not account
for the SR-AI/II-independent uptake of oxidized LDL in murine
macrophages. However, in human macrophages, anti-CD36 antibody has been
shown to partly inhibit the degradation of oxidized LDL (50, 51), and
monocyte-macrophages from subjects with inherited deficiency of CD36
were found to have a reduced rate of uptake and degradation of oxidized
LDL (52). Hence, it is possible that CD36 is a mediator of oxidized LDL
uptake and degradation in human cells. Acton et al. (53) identified a scavenger receptor (termed SR-BI) homologous to CD36 that
binds oxidized or acetylated LDL but also native LDL (53). It also
binds high density lipoprotein, and it has been proposed that one
function of SR-BI may be to promote adhesion of high density
lipoprotein to cells and facilitate cholesterol ester transfer (54).
More recently, a 94-97-kDa macrophage plasma membrane protein
identified by ligand blotting has been proposed to be a specific
receptor for oxidized LDL (55, 56). This protein has been identified as
macrosialin, the murine homologue of CD68 (57). Although macrosialin is
targeted to lysosomes and only a small percentage of the total appears
on the plasma membrane, it is so abundantly expressed in macrophages
that a significant number of molecules are detectable on the cell
surface (58). Antibodies to CD68 partially inhibited the uptake of
oxidized LDL by THP-1 cells (58).
Evaluation of the role of these candidate receptors for oxidized LDL is rendered difficult because of the presence of scavenger receptors in macrophages, because any effect of these postulated "specific" receptors for oxidized LDL is observed against a high background level of uptake via the SR-AI/II. To date, no normal cell type has been identified that expresses oxidized LDL receptors and not SR-AI/II. However, it has been suggested that Kupffer cells may exhibit a relatively high level of "oxidized LDL receptor" activity. Nevertheless, these cells also have scavenger receptors, and the results of competition experiments remain somewhat equivocal. Because of this "interference" by scavenger receptors, identification of specific oxidized LDL receptors would be greatly facilitated in animals where scavenger receptors were absent. Suzuki et al. (59) have recently generated transgenic mice in which the SR-AI/II (msr) gene was inactivated by targeted gene disruption. The homozygous mutants have complete absence of SR-AI/II protein, but their phenotype is virtually normal. The present studies were done to characterize the uptake and degradation of modified lipoproteins by macrophages from these SR-AI/II-deficient animals.
Carrier-free 125I and 32P were purchased
from Mandel Scientific (Guelph, Ontario, Canada). Gentamicin,
-minimal essential medium, ultra pure agarose, proteinase K,
ethidium bromide, BglII, XbaI, 1-kb DNA ladder,
and high molecular weight DNA markers were from Canadian Life
Technologies (Burlington, Ontario, Canada). Hyclone-defined fetal
bovine serum and Stratagene NucTrap probe purification columns were
purchased from Professional Diagnostics (Edmonton, Alberta, Canada).
Arachidonic acid, linoleic acid, bovine serum albumin, n-octyl
-D-glucopyranoside, phorbol myristate
acetate, cholesterol, tetramethoxypropane and phenylmethylsulfonyl
fluoride were obtained from Sigma. Phosphatidylserine and
phosphatidylcholine were from Avanti Polar Lipids (Birmingham, AL).
Nitrocellulose membrane and prestained SDS-polyacrylamide gel
electrophoresis standards were purchased from Bio-Rad. Hybond-N
hybridization transfer membrane was obtained from Amersham Corp. Kodak
X-Omat AR film was from MedTec Marketing (Richmond, British Columbia,
Canada). Other reagents were purchased from Fisher Scientific
(Vancouver, British Columbia, Canada) or VWR Canlab (Edmonton, Alberta,
Canada).
The procedure for disruption of the msr gene (which encodes SR-AI/II) has been reported recently (59). A targeting vector was introduced into exon 4 (the ligand-binding region) of the gene. Brother-sister mating of heterozygotes was carried out to generate homozygous mutants. Normal littermates were bred as controls. To verify that there had been no accidental intermingling of wild-type and homozygous mice during the study, genotyping of selected experimental animals throughout the study as well as of all breeders was performed by Southern blotting using a 600-base pair probe from a region adjacent to exon 4 of the msr gene. Digestion of genomic DNA with BglII generated a single 23-kb band in wild-type mice and a single 10-kb band in homozygous knockout mice. None of the mice showed both the 23-kb band and the 10-kb band characteristic of heterozygotes.
Lipoprotein Isolation and LabelingLDL (d = 1.019 1.063) was isolated by sequential ultracentrifugation of
EDTA-anticoagulated fasting plasma obtained from healthy normolipidemic
volunteers (60). Radioiodination was performed using a modification of
the iodine monochloride method of MacFarlane (61). Specific
radioactivities were 100-250 cpm/ng. Iodination was performed prior to
modification of LDL.
The concentration of EDTA in LDL preparations was reduced prior to oxidation by dialysis against Dulbecco's phosphate-buffered saline containing 10 µM EDTA. Standard conditions for LDL oxidation were: 200 µg/ml LDL in Dulbecco's phosphate-buffered saline containing 5 µM CuSO4 incubated at 37 °C for 20 h (62). This typically resulted in electrophoretic mobility of 0.85 relative to albumin. For some experiments, the extent of oxidation was controlled by varying the incubation time. Mildly oxidized LDL was prepared by incubating for 3.5 h and resulted in electrophoretic mobility 0.45 relative to albumin. Extensively oxidized LDL was prepared by incubating for 30 h and resulted in electrophoretic mobility of 1.06 relative to albumin. Acetylation, malondialdehyde modification, arachidonic acid oxidation product modification or linoleic acid oxidation product modification of LDL, or bovine serum albumin was performed as described previously (63).
Assays with Cultured MacrophagesResident peritoneal
macrophages were obtained from wild-type mice or SR-AI/II knockout mice
by peritoneal lavage with ice-cold Ca2+-free Dulbecco's
phosphate-buffered saline. Cells were suspended in -minimal
essential medium with 10% fetal bovine serum and plated in 12-well
plastic culture plates at a density of 1 × 106
cells/well. Adherent macrophages were cultured overnight in a humidified CO2 incubator and then washed with serum-free
-minimal essential medium. Modified LDLs with or without competitors
were added to the cells in
-minimal essential medium supplemented with 2.5 mg/ml lipoprotein-deficient serum to minimize cytotoxicity. For studies with polycytidylic acid, 10 units/ml recombinant
ribonuclease inhibitor (Promega Corp.) was added to prevent degradation
of this polynucleotide (43). After 5 h incubation at 37 °C,
media were removed and assayed for trichloroacetic acid-soluble
noniodide degradation products. Cells were washed twice with
Dulbecco's phosphate-buffered saline, dissolved in 0.1 N
NaOH, scraped from the plates, counted, and assayed for protein
content.
To obtain phosphatidylserine liposomes, 12 µmol of bovine brain phosphatidylserine, 12 µmol of egg phosphatidylcholine, and 12 µmol of cholesterol were suspended in 1.5 ml of 150 mM NaCl, 10 mM Hepes, and 0.1 mM EDTA, pH 7.5. The mixture was passed 10 times though a 0.1-µm polycarbonate membrane using a Lipex mini-extruder. Phosphatidylcholine liposomes were prepared with the same procedure, except that 20 µmol of egg phosphatidylcholine and 10 µmol of cholesterol were used.
Macrophage Membrane PreparationResident peritoneal macrophages from 35-40 mice were suspended in cold 5 mM Tris-HCl, pH 8.0, containing 0.25 M sucrose and 1 mM phenylmethylsulfonyl fluoride and disrupted by two cycles of nitrogen cavitation at 460 kPa for 30 min. Nuclei were pelleted by centrifugation at 1000 × g for 10 min at 4 °C. The suspension was centrifuged at 10,000 × g for 15 min, and the resulting supernatant was centrifuged at 100,000 × g for 120 min at 4 °C. The crude membrane pellet was solubilized in 100 µl of 10 mg/ml octyl glucoside containing 1 mM phenylmethylsulfonyl fluoride and 0.1 mM EDTA and assayed for protein content. Typical yields of macrophage membrane protein were 0.3-1 mg. Solubilized membrane was stored at 4 °C and used within 1 day for ligand blotting.
Ligand BlottingLigand blotting was performed according to
Ottnad et al. (55). Crude membrane proteins were
electrophoresed in SDS/8% polyacrylamide mini gels under nonreducing
conditions and electroblotted onto nitrocellulose membranes. Membrane
strips were preincubated for 1 h with 5% nonfat dry milk in 10 mM Tris-HCl, 90 mM NaCl, and 1 mM
EDTA, and then 10 µg/ml of native or modified
125I-labeled LDL (150-250 cpm/ng) were added for an
additional 1.5 h. The nitrocellulose strips were washed eight
times for 10 min with buffer containing 1% nonfat dry milk, air-dried,
and exposed to Kodak X-Omat AR film for 10 days at 70 °C.
Protein determination was done by the method of Lowry et al. (64) in the presence of 0.05% sodium deoxycholate to minimize turbidity. Bovine serum albumin was used as the standard. Lipoprotein electrophoresis was done using a Corning apparatus and Universal agarose film in 50 mM barbital buffer, pH 8.6. Lipoprotein bands were visualized by staining with fat red.
Assuming that the expression of other receptors is not altered by
the disruption of the SR-AI/II gene, then the uptake of acetyl LDL or
oxidized LDL by macrophages via "alternate" receptors should be
equivalent to saturable uptake in macrophages from knockout mice,
whereas the uptake via scavenger receptors should be the difference in
uptake between wild-type and knockout macrophages. Accordingly,
resident peritoneal macrophages from wild-type mice or scavenger
receptor knockout mice were incubated with increasing concentrations of
125I-labeled acetyl LDL or 125I-labeled
oxidized LDL. As shown in Fig. 1, the uptake and
degradation of acetyl LDL was reduced by more than 80% in macrophages
from scavenger receptor knockout mice. This is direct evidence that most of the uptake of acetyl LDL by macrophages can be attributed to
the SR-AI/II. In contrast, the uptake and degradation of extensively oxidized LDL was reduced by only 30% in knockout mice and showed high
affinity, saturable kinetics with an apparent Km of
about 5 µg/ml, similar to that of SR-AI/II. This suggests that about
70% of the uptake of oxidized LDL in macrophages is attributable to an
alternate oxidized LDL receptor(s). Fig. 1 also shows that after 5 h incubation with acetyl LDL, the amount of cell-associated LDL was
about 20% of the amount degraded, whereas with oxidized LDL, this was
similar to or even greater than the amount degraded. This is not
surprising, because oxidized LDL has been shown previously to resist
lysosomal degradation, and hence tends to accumulate within cells (65,
66).
To define the degree of oxidative modification of LDL that is required
for high affinity uptake in SR-AI/II knockout macrophages, the uptake
of LDL preparations that differed in degree of oxidation was compared.
Results shown in Fig. 2 indicate that the uptake of
mildly oxidized LDL was the same as native LDL, and that at least a
moderate degree of oxidation is required for recognition by the
alternate receptor(s). This oxidized LDL had electrophoretic mobility
3.4-fold that of native LDL, which corresponds to derivatization of
about 40% of lysine residues (35). This degree of modification is
similar to the "threshold" degree of modification required for
uptake of oxidized or chemically modified LDL by the SR-AI/II (35,
67).
Scavenger receptors have a rather broad and often overlapping ligand
specificity but can be distinguished on the basis of binding to
polynucleotides in that class A receptors are capable of interacting
with polyinosinic acid and certain other polynucleotides, whereas class
B receptors do not (53). To further characterize the uptake pathway for
oxidized LDL in SR-AI/II knockout macrophages, several different
modified LDLs and polynucleotides were tested for their ability to
compete for the degradation of radiolabeled oxidized LDL. Fig.
3 shows that as expected, unlabeled oxidized LDL was an
effective competitor, whereas acetyl LDL was a poor competitor.
Polyinosinic acid completely blocked oxidized LDL uptake, indicating
that the uptake of oxidized LDL in knockout macrophages cannot be
attributed to SR-B. We have shown previously that reaction of LDL with
fatty acid peroxidation products (under conditions where oxidation of
LDL itself was prevented) results in high affinity saturable uptake by
macrophages (63, 68). To determine if oxidation product-modified LDL
was a ligand for the "alternate" scavenger receptor in SR-AI/II
knockout macrophages, we tested its ability to compete for the uptake
of oxidized LDL in these cells. As shown in Fig.
4A, LDL that had been modified by arachidonic
acid oxidation products competed for the uptake of oxidized LDL, and
the efficiency of oxidation product-modified LDL depended on its extent
of derivatization as reflected by electrophoretic mobility. Bovine
serum albumin modified by arachidonic acid oxidation products was also
a good competitor, indicating that neither apolipoprotein B nor
phospholipids were required for ligand activity. Similar results were
obtained when oxidation products from linoleic acid were used to modify
LDL, except that a much lower degree of modification (as judged by
electrophoretic mobility in agarose) was required to fully compete for
oxidized LDL uptake. This suggests that this receptor can distinguish
between different lipid peroxidation products, and that those derived
from linoleic acid may generate a higher affinity ligand.
Although many potentially reactive products have been found after
autooxidation of polyunsaturated fatty acids, 2-unsaturated aldehydes
and malondialdehyde are thought to be among the more important (69). To
assess the specificity of the alternate receptor(s) for
aldehyde-modified proteins, malondialdehyde and simple 2-unsaturated aldehydes of varying chain length were used to modify LDL, and the
resulting modified LDLs were tested for their ability to compete for
the degradation of radioiodinated oxidized LDL by macrophages from
SR-AI/II knockout mice. Fig. 5A shows that
acrolein-modified LDL and malondialdehyde-modified LDL failed to
compete for oxidized LDL uptake. Heptenal-LDL was a moderately
effective competitor but was much less potent than LDL modified by
fatty acid peroxidation products. This suggests that the lipid
peroxidation products that cause binding to the alternate receptor(s)
are unlikely to be simple short-chain aldehydes. Several scavenger
receptors, including macrosialin, CD36, and SR-BI are reported to
interact with phosphatidylserine-rich membranes (57, 70). To determine
if the uptake of oxidized LDL in SR-AI/II-deficient macrophages might
involve one of these receptors, we tested the effect of
phosphatidylserine-rich liposomes on oxidized LDL degradation. As shown
in Fig. 5B, phosphatidylserine liposomes were effective
competitors, whereas phosphatidylcholine liposomes had no effect.
As a preliminary step to identifying membrane proteins that might
mediate the binding of oxidized LDL in the SR-AI/II knockout animals,
we separated membrane proteins from knockout macrophages by
SDS-polyacrylamide gel electrophoresis, transferred these to nitrocellulose membranes, and blotted the membranes with radioiodinated extensively oxidized LDL, mildly oxidized LDL, native LDL, or acetyl
LDL. As shown in Fig. 6, the pattern of bands was
similar with all lipoproteins; the two most intense were at 94 and 210 kDa, with minor bands at 60 and 124 kDa. Binding to the 60-, 94-, and
210-kDa bands was completely blocked by polyinosinic acid. The same
bands were seen in membrane proteins from control macrophages, but in
addition, a band at 220-240 kDa was visualized with acetyl LDL that
probably represents the trimeric SR-AI. The 94-kDa band has been
identified as macrosialin, and according to Ramprasad et al.
(57), the 210-kDa band may be a dimer of macrosialin. The identity of
the other bands is at present unknown. The finding that native LDL
interacted on ligand blotting with a protein at 94 kDa, whereas native
LDL had no effect on the rate of oxidized LDL uptake, would suggest
that this band is not the receptor. However, SR-BI is known to bind LDL
and, in its glycosylated form, is similar in size to macrosialin;
hence, the band at 94 kDa seen with native LDL might represent SR-BI
rather than macrosialin. Because macrosialin is said to be up-regulated
in thioglycollate-elicited macrophages (57), we compared the uptake of
oxidized LDL in elicited macrophages from knockout mice with that in
resident macrophages and found no increase in the elicited macrophages. We also tested the effect of preincubating macrophages from knockout mice with 15-100 nM phorbol myristate acetate, which is
reported to increase cell-surface macrosialin in THP-1 cells (58), but found no increase in the rate of oxidized LDL uptake (data not shown).
However, these negative results do not exclude a possible role for
macrosialin in the uptake of oxidized LDL in these cells because we
were unable to obtain an antibody to murine macrosialin and hence could
not verify that these interventions had actually increased macrosialin
expression.
The existence of a specific receptor(s) for oxidized LDL was initially proposed on the basis of studies showing incomplete competition by acetyl LDL for the uptake and degradation of oxidized LDL (44, 45, 49). However, the extent of competition of acetyl LDL for oxidized LDL uptake depends greatly on the extent of oxidation and aggregation of oxidized LDL, and hence it is difficult to assess the relative importance of this "specific" receptor(s) in the uptake of oxidized LDL compared with uptake via the SR-AI/II (71). The findings in the present study with SR-AI/II knockout mice provide direct evidence confirming the existence of a separate, high affinity saturable pathway for the uptake and degradation of oxidized LDL. Furthermore, the results indicate that this pathway accounts for about 70% of the uptake of extensively oxidized LDL in murine macrophages, and that only 30% is attributable to SR-AI/II. These findings differ significantly from those reported recently by Sakai et al. (72), who found that the degradation of oxidized LDL was decreased by 70% in SR-AI/II macrophages. This difference is probably a result of differences in the degree of LDL oxidation and in the assay conditions. For instance, the rate of uptake in our studies was an order of magnitude greater than that of Sakai's, suggesting that the extent of oxidation in their studies may not have been sufficient to obtain an optimal ligand. Competition studies in knockout mice revealed some characteristics in common with the SR-AI/II, in that extensive LDL oxidation was required for uptake, and polyinosinic acid was a good competitor for oxidized LDL uptake whereas polycytidylic acid, which does not form a base-quartet-stabilized helix (43), was ineffective. The ligand specificity of the uptake pathway in knockout mice differed from that of the SR-AI/II in that the apparent affinity of this alternate pathway toward acetyl LDL and malondialdehyde-modified LDL was at least an order of magnitude lower than that for oxidized LDL.
Several macrophage membrane proteins that bind oxidized LDL have
recently been identified including FcRII-B2 (49), CD36 (50), SR-BI
(54), and macrosialin, the mouse homologue of human CD68 (57). Recent
studies have shown that SR-BI binds native LDL with high affinity and
that both SR-B1 and CD36 are unable to bind to polyanionic ligands such
as poly I (54). In addition, CD36 binds mildly oxidized LDL at least as
well as extensively oxidized LDL (50). These characteristics are
clearly different from those associated with oxidized LDL uptake in
knockout macrophages, indicating that neither of these class B
scavenger receptors are likely to be responsible for oxidized LDL
uptake in these cells. However, there is evidence that CD36 may play a
role in human macrophages, in that monocyte-derived macrophages from
CD36-deficient subjects have about a 50% reduction in binding and
uptake of oxidized LDL, and anti-CD36 blocks 22-50% of oxidized LDL
binding and uptake in normal human macrophages (50-52).
Macrosialin/CD68 has recently been proposed as a receptor for oxidized LDL (57). The interaction of oxidized LDL with macrosialin/CD68 has been characterized mostly on the basis of ligand blot analyses (55, 57). However, the ligand specificity appears strikingly similar to that we found for the uptake of oxidized LDL by SR-AI/II knockout macrophages in that there is no interaction with native LDL, a higher affinity for oxidized LDL than acetyl LDL (55), inhibition of ligand binding with polyinosinic acid but not polycytidylic acid, and binding with phosphatidylserine-rich but not with phosphatidylcholine liposomes. This concordant ligand specificity is consistent with the hypothesis that macrosialin may be an important mediator of oxidized LDL uptake. We found no increase in oxidized LDL uptake with thioglycollate-elicited macrophages or with cells pretreated with phorbol ester (both of which are expected to increase cell-surface macrosialin), but Ramprasad et al. (58) showed recently that antibodies to CD68 specifically inhibited about 20% of the uptake of oxidized LDL by human THP-1 macrophage-like cells. Definitive assessment of the role of macrosialin/CD68 may require inhibiting its expression by targeted gene disruption. Nishikawa et al. (73) described a receptor for acidic phospholipid vesicles that shares many properties with macrosialin and may in fact be the same protein (74, 75).
Elomaa et al. (76) have described a new receptor structurally similar to the SR-AI that is expressed only on macrophages in the marginal zone of the spleen and in the medullary cord of lymph nodes. It binds acetyl LDL, but its relative affinity for oxidized LDL and the level of expression by peritoneal macrophages have not yet been reported. This receptor is not detectable in macrophages from lung or liver, and hence its tissue distribution cannot account for the fact that in SR-AI/II knockout animals, 60-80% of intravenously injected oxidized LDL or acetyl LDL is cleared by the liver, whereas only about 1% is taken up by the spleen.2
A number of other scavenger receptors have been described, including a 125-kDa glycoprotein that binds glycosylated or aldehyde-modified proteins (77, 78), a 90-kDa receptor for proteins modified by advanced glycosylation end-products (79), receptors for conformationally modified albumins (80-83), and a "novel" acetyl-LDL receptor (84). Although not all of these receptors have been fully characterized, none has a ligand specificity that is the same as that of the uptake pathway for oxidized LDL in SR-AI/II knockout macrophages.