(Received for publication, February 19, 1996)
From the
Several investigators have reported nonreciprocal
cross-competition between unlabeled acetyl low density lipoprotein
(LDL) and oxidized LDL for the degradation of the corresponding labeled
LDLs. The failure of acetyl LDL to compete fully for the degradation of
oxidized LDL has been interpreted as evidence for additional
receptor(s) specific for oxidized LDL. In the present study, it is
demonstrated that the ability of oxidized LDL to compete for the
degradation of acetyl LDL is determined largely by its extent of
oxidation. Extensively oxidized LDL competed for 90% of acetyl LDL
degradation in murine macrophages, and hence there appears to be no
pathway in these cells that is specific for acetyl LDL but not oxidized
LDL. The reciprocal situation (competition by acetyl LDL for uptake and
degradation of oxidized LDL) proved to be more complicated. Oxidized
LDL is known to be susceptible to aggregation, and less than half of
the aggregates found in the present experiments were large enough to be
removed by filtration or centrifugation at 10,000 g.
When oxidized LDL was prepared under conditions that resulted in
minimal aggregation, acetyl LDL competed for greater than 80% of
oxidized LDL degradation. With more extensive oxidation and aggregation
of LDL, acetyl LDL only competed for about 45% of oxidized LDL
degradation, while polyinosinic acid remained an effective competitor.
Individual preparations of oxidized LDL that differed in degree of
oxidation were separated into aggregated and nonaggregated fractions,
and it was shown that both fractions were competed to a similar degree
by acetyl LDL in mouse peritoneal macrophages and in Chinese hamster
ovary cells transfected with human scavenger receptor type I cDNA.
Hence, aggregation by itself did not alter the apparent rate of uptake
by the scavenger receptor pathway. These results indicate that the
extent of oxidation of LDL affects its mechanism of uptake and that
about half of the uptake of very extensively oxidized LDL appears to be
via a pathway distinct from the scavenger receptor type I/II. The
uptake of very extensively oxidized LDL was not affected by
cytochalasin D, an inhibitor of phagocytosis. As well, it was not
affected by an antibody to CD36 in human monocyte-derived macrophages
or in THP-1 cells, suggesting that this alternate pathway does not
involve CD36.
Oxidatively modified LDL ()has been shown to have
many potentially atherogenic actions and properties (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) .
There is considerable evidence that at least a mild degree of oxidation
of LDL occurs in arterial lesions in vivo(13, 14, 15, 16, 17) .
The possibility that oxidation of LDL might play a causal role in
atherogenesis was suggested by several studies showing that antioxidant
drugs can slow the progression of atherosclerosis in experimental
animals(18, 19, 20, 21) .
One of the properties associated with extensively oxidized LDL is the ability to interact with scavenger receptors on phagocytic cells(22, 23) . It has been postulated that this could lead to unregulated delivery of LDL to macrophages, resulting in the formation of foam cells(1) . Scavenger receptor cDNA of bovine, murine, rabbit, and human origin have been cloned and sequenced(24, 25, 26, 27, 28, 29, 30) . The scavenger receptor has six structural domains, including a collagen-like domain (V) that is believed to mediate ligand binding. The human scavenger receptor gene is located on chromosome 8, and two forms of the receptor (termed type I and type II) are produced from this gene through alternate splicing of mRNA(31) . The cysteine-rich extracellular domain VI is deleted in the type II receptor, without evident effect on binding of most ligands(32) , although recently it has been shown that bacterial lipopolysaccharide binds less well to type I than to type II receptors(30) . Various substances that are structurally unrelated except that they have domains with high negative charge bind to the receptor, including polyinosinic acid, fucoidan, dextran sulfate, maleylated albumin, and acetyl LDL(33) . This suggests that ligand binding may be mediated principally by ionic interactions. Freeman and co-workers (28) found that although both acetyl LDL and oxidized LDL were internalized and degraded by Chinese hamster ovary cells transfected with type I or type II bovine scavenger receptors, and acetyl LDL competed efficiently for degradation of oxidized LDL, oxidized LDL did not compete well for acetyl LDL degradation. Very similar results were reported by Dejager and colleagues (34) in both scavenger receptor-transfected Chinese hamster ovary cells and phorbol ester-treated rabbit smooth muscle cells. It was proposed that the failure of oxidized LDL to compete for acetyl LDL degradation might reflect nonidentical binding sites for the two ligands on the same receptor molecule(28) . Additional evidence for this was provided by Doi and colleagues(35) , who analyzed the effect of deletion mutations on the human type I scavenger receptor, and concluded that a cluster of lysine residues within the C-terminal 22 amino acids of the collagen-like domain of the receptor were essential for ligand binding. There were differences in sensitivity to individual lysine mutations between acetyl LDL and oxidized LDL, consistent with the notion that the binding sites for these ligands may not be identical.
Several groups have reported
evidence of a receptor for oxidatively modified LDL distinct from the
scavenger receptor on macrophages (36, 37, 38) or Kupffer
cells(39, 40, 41) . 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(36, 37, 39, 40) . 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(39) . However, there are several potential
explanations for this observation, including the possibility that
aggregation affecting only the oxidized LDL preparations in this study
might have caused increased Kupffer cell uptake. Endemann and
colleagues (38, 42) undertook the identification of
new receptors for oxidized LDL using a mouse cDNA library and an
expression cloning strategy with fluorescent-labeled oxidized LDL. They
identified 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 FcRII-B2 receptor(38) . However, blocking
antibodies to the Fc
RII-B2 receptor failed to inhibit the uptake
of oxidized LDL in mouse peritoneal macrophages, and hence it is
questionable if this receptor accounts for a significant component of
oxidized LDL uptake in these cells. The second protein was shown to be
the murine homologue of CD36(42) . Studies in 293 cells
transiently transfected with CD36 showed an increase in binding of
oxidized LDL, but degradation was very low, and hence it was unclear
from this experiment if CD36 could account for the scavenger
receptor-independent uptake and degradation of oxidized
LDL(42) . It was found that only a mild degree of oxidation of
LDL was sufficient to permit binding to this protein, in contrast to
the extensive oxidation of LDL required for receptor-mediated uptake
and degradation in mouse peritoneal macrophages(23) .
Monoclonal antibody OKM5 against human CD36 reduced the binding of
oxidized LDL to THP-1 human monocytic leukemia cells by 52%, but the
effect of this antibody on LDL degradation was not reported. More
recently, Nicholson and colleagues (43) reported that anti-CD36
antibody 8A6 inhibited the degradation of oxidized LDL by human
monocyte-macrophages by 22%. More direct evidence for a role for CD36
in oxidized LDL uptake was the finding that monocyte-macrophages from
subjects with inherited deficiency of CD36 have a reduced rate of
uptake and degradation of oxidized LDL(44) . Hence, it is
possible that CD36 is a mediator of oxidized LDL uptake and
degradation, at least in human macrophages.
There are a number of issues that require clarification before the results in the above studies can be reconciled. In particular, an explanation is needed for the finding in some studies that acetyl or acetoacetylated LDL competed fully for oxidized LDL uptake and degradation in macrophages(23, 34, 41) , while in others it competed only partially(36, 38, 40) . The failure of acetyl LDL to compete for oxidized LDL degradation in macrophages cannot adequately be accounted for simply on the basis of nonidentical binding sites on scavenger receptors type I or II for these two ligands, because acetyl LDL was found to compete well for oxidized LDL degradation in cells expressing only type I or type II scavenger receptors(28, 34) .
The objectives of the present study were to determine if the uptake of oxidized LDL by macrophages involved one or more than one major pathway, and if the apparent non-reciprocal cross-competition between oxidized LDL and acetyl LDL (and some of the discrepancies between previous publications) might be due to ligand heterogeneity of oxidized LDL rather than heterogeneity or multiplicity of receptors. Specifically, we wished to determine if heterogeneity in terms of the extent of oxidation of LDL and/or the degree of aggregation in oxidized LDL might account for these findings.
Carrier-free I was purchased from DuPont NEN
(Lachine, Quebec, Canada).
-Minimal essential medium, fetal bovine
serum, and gentamicin were from Life Technologies, Inc. (Burlington,
Ontario, Canada). Monoclonal antibody to human CD36 (CLB-703) was
purchased from Cedarlane Laboratories, Hornby, Ontario, Canada.
Purified human platelet thrombospondin was a gift from Dr. Dana Devine,
Department of of Pathology, University of British Columbia. Female CD-1
mice were supplied by the University of British Columbia Animal Care
Centre. Formaldehyde was from J. B. EM Services (Dorval, Quebec,
Canada). Butylated hydroxytoluene was purchased from J.T. Baker
(Toronto, Ontario, Canada). DiI,
(1,1`-dioctadecyl-3,3,3`,3`-tetramethylindocarbocyanine perchlorate)
was obtained from Molecular Probes (Eugene, OR). Other chemicals and
solvents were purchased from Fisher Scientific (Vancouver, British
Columbia, Canada) or BDH (Toronto).
Human monocytes were isolated from freshly
obtained citrate-anticoagulated blood using Ficoll-Hypaque. Multiple
tubes containing 30 ml of blood layered over 15 ml of Ficoll-Hypaque
were centrifuged at 440 g for 25 min at 10 °C, and
the mononuclear cells at the interface were collected and pooled.
Platelets were removed by differential centrifugation in RPMI 1640.
Purified mononuclear cells were added to 10-cm diameter Costar plastic
culture dishes at a density of 2
10
cells/ml and
incubated undisturbed for 1 h at 37 °C. Nonadherent cells were then
removed by three washes with prewarmed RPMI 1640 medium with 100
units/ml penicillin and 100 µg/ml streptomycin. Loosely adherent
monocytes were scraped off in cold RPMI with a sterile cell lifter and
counted. The viability was >95% as judged by trypan blue exclusion.
To permit differentiation to macrophages, 1-2
10
monocytes/well were plated in 24-well culture plates with 1 ml of
RPMI 1640 containing 20% autologous serum. Cells were used for
experiments between 9 and 12 days after seeding. For each experiment
only cells derived from one donor were used. Autologous serum was
prepared from blood that had been allowed to clot at 37 °C for 2 h
by centrifugation at 3200
g for 15 min at room
temperature. The differentiation of monocytes into macrophages under
these conditions was assessed by morphology and level of expression of
scavenger receptor activity, which increased 10-fold as judged by
uptake and degradation of acetyl LDL. THP-1 human monocytic leukemia
cells were obtained from American Type Culture Collection (Rockwell,
MD) and were cultured in RPMI 1640 medium with 10% FBS. THP-1 cells
were induced to differentiate to a macrophage-like phenotype by 72 h of
exposure to 200 ng/ml phorbol myristate acetate.
It has been proposed that macrophages possess at least two
pathways for the uptake of oxidized LDL: one shared with acetyl LDL and
mediated by scavenger receptors type I and II, and another pathway or
pathways specific for oxidized LDL but not acetyl LDL(36) . If
this were the case, one would expect that oxidized LDL would compete
fully for the degradation of acetyl LDL, but that acetyl LDL would
compete for only part of the degradation of oxidized LDL. However,
depending on the conditions employed, oxidized LDL preparations can
differ greatly in their extent of oxidation and degree of aggregation
as well as in the nature of lipid peroxidation products present, and
the degree of derivatization and fragmentation of apoB. To determine
how the extent of oxidation affects binding to the acetyl LDL scavenger
receptor, a series of oxidized LDL preparations that differed in their
extent of oxidation were compared for their ability to compete for the
degradation of I-acetyl LDL. It should be noted that all
of these oxidized LDL preparations were modified to an extent
sufficient for rapid high affinity uptake in macrophages (the
``threshold'' level of oxidation for scavenger receptor
recognition typically occurs at electrophoretic mobility
0.70-0.75 relative to albumin). Results shown in Fig. 1A demonstrate that the effectiveness of oxidized
LDL as a competitor for acetyl LDL degradation varies greatly depending
on the extent of oxidation. The most extensively oxidized LDL
preparation in this experiment competed for about 90% of acetyl LDL
degradation, although the apparent affinity was less than that of
unlabeled acetyl LDL. Thus, in contrast to a report by Arai and
colleagues(37) , in mouse peritoneal macrophages we found no
evidence of a specific receptor for acetyl LDL that did not recognize
oxidized LDL. Although electrophoretic mobility is a convenient and
reproducible indicator of the extent of LDL oxidation by copper, it is
important to standardize this against a quantitative measure of the
amount of fatty acid substrate consumed by lipid peroxidation.
Accordingly, LDL samples with varying degrees of oxidation were
analyzed for fatty acid composition. Arachidonic acid was consumed very
rapidly, but trace amounts were difficult to quantify because oxidation
generated many new peaks in that region of the chromatogram. As
expected, oleic acid was relatively stable, with only 23% consumption
even with extensively oxidized LDL. Fig. 1B describes
the correlation between electrophoretic mobility and consumption of
linoleic acid (the most abundant unsaturated fatty acid in LDL). Nearly
all of the linoleic acid was consumed in LDL with electrophoretic
mobility greater than 0.9, indicating that these samples were maximally
oxidized.
Figure 1:
Effect of extent of
oxidation on the ability of oxidized LDL to compete for acetyl LDL
degradation. A, macrophages were incubated 5 h with 5
µg/ml acetyl I-LDL together with varying
concentrations of unlabeled native LDL (
, electrophoretic mobility
0.22); unlabeled LDL oxidized by exposure to 5 µM Cu
for 18 h (
, electrophoretic mobility
0.83), 21 h (
, electrophoretic mobility 0.91), 28 h (
,
electrophoretic mobility 0.98), 30 h (
, electrophoretic mobility
1.02), or unlabeled acetyl LDL (
). Acetyl LDL degradation is
expressed as a percentage of that in the absence of competitor. Values
shown are means of duplicate incubations that varied by less than 10%. B, fatty acid composition was determined for native LDL
(
) and LDL oxidized to varying degrees as above (
). The
percentage of linoleic acid consumed is plotted as a function of
electrophoretic mobility of LDL.
Hoppe and colleagues (53) have reported that oxidized LDL can inactivate lysosomal proteases, and hence the observed inhibition of degradation of acetyl LDL might be due to lysosomal dysfunction rather than to competition for receptors. If this were the case, one would expect to find intracellular accumulation of radioactivity from internalized but undegraded acetyl LDL in the presence of oxidized LDL. To test this possibility, cell-associated radioactivity was measured in parallel with degradation. Even at the highest concentration of oxidized LDL, cell-associated acetyl LDL radioactivity was less than 5% of the amount degraded in the absence of competitor, and therefore most of the inhibition of degradation of acetyl LDL by oxidized LDL was due to competition for uptake and not to lysosomal dysfunction caused by oxidized LDL.
The next experiments
were done to determine why acetyl LDL sometimes fails to compete fully
for the degradation of oxidized LDL. Fig. 2shows a typical
experiment comparing the ability of acetyl LDL and oxidized LDL to
compete for the uptake and degradation of I-oxidized LDL.
The abrupt drop and subsequent plateau of the competition curve with
acetyl LDL suggests that there is more than one class of
ligand-receptor interaction between oxidized LDL and the cells, only
one of which is efficiently competed by acetyl LDL. Similar results
have been reported previously (36) and, as noted above, have
been taken as evidence for more than one receptor. However, this
finding could also be explained by heterogeneity of the labeled
oxidized LDL ligand. A likely source of heterogeneity in oxidized LDL
is aggregation, because oxidized LDL is known to be very susceptible to
aggregation(54) . To test whether aggregation of oxidized LDL
might account for the failure of acetyl LDL to compete completely for
oxidized LDL uptake, we generated labeled oxidized LDL preparations
with differing levels of aggregation and compared the ability of acetyl
LDL to compete for their uptake. The extent of aggregation of oxidized
LDL was varied in three different ways: by changing the extent of
oxidation, by briefly vortexing a ``standard'' oxidized LDL
preparation, or by increasing the concentration of LDL during
oxidation. The extent of aggregation was estimated by agarose gel
electrophoresis and by centrifugation at 10,000
g (Table 1, Fig. 3). With electrophoresis, aggregation
was calculated as the amount of radioactivity recovered in the gel lane
from the origin to the lower edge of the main LDL band divided by total
radioactivity in that lane. This method demonstrated aggregates that
were evidently too small to sediment at 10,000
g.
Results shown in Fig. 4indicate that acetyl LDL competed for
more than 80% of the degradation of ``standard'' oxidized LDL
(11% aggregates by electrophoresis) but only about 50% of the
degradation of vortexed oxidized LDL (36% aggregates by
electrophoresis). The extent of aggregation of oxidized LDL as assessed
by electrophoresis correlated with the proportion of oxidized LDL
degradation that was unaffected by unlabeled acetyl LDL. This result
suggests that aggregation of oxidized LDL might account for the failure
of acetyl LDL to compete fully for oxidized LDL degradation in mouse
peritoneal macrophages.
Figure 2:
Incomplete competition by acetyl LDL for
uptake of oxidized LDL. Macrophages were incubated for 5 h with 3
µg/ml oxidized I-LDL (relative electrophoretic
mobility 0.95) together with the indicated concentration of unlabeled
native LDL (
), acetyl LDL (
), malondialdehyde-modified LDL
(
), or oxidized LDL (
). The amount of cell-associated and
degraded LDL was determined as described in the methods. Each point is
the mean of duplicates that varied by less than 10%. The value for
oxidized LDL in the absence of competitor was 18.1 µg/mg. Similar
results were obtained in five of five such
experiments.
Figure 3:
Agarose gel electrophoresis. Oil red
O-stained gel showing native LDL (lane 1),
``standard'' oxidized LDL (lane 2), vortexed
oxidized LDL (lane 3), LDL oxidized by incubating a high
concentration of LDL (2 mg/ml) with 20 µM Cu for 40 h at 37 °C (lane 4), LDL very extensively
oxidized by incubating 200 µg/ml LDL with 5 µM Cu
for 30 h (lane 5), and acetyl LDL (lane 6). The arrow indicates the
origin.
Figure 4:
Effect of varying the extent of
aggregation of oxidized I-LDL on the ability of acetyl
LDL to compete for its degradation. ``Standard'' oxidized
I-LDL (
) or oxidized
I-LDL with
aggregation induced by vortexing (
), very extensive oxidation
(
,
), or oxidation at high LDL concentrations (
)
was incubated with macrophages for 5 h in the presence of the indicated
concentration of acetyl LDL (
,
,
,
) or
polyinosinic acid (
). Characterization of these oxidized LDL
preparations is shown in Table 1. Oxidized LDL degradation is
expressed as a percentage of that in the absence of competitor. Values
are means of duplicate incubations that varied by less than 10%. The
results shown are representative of three experiments. Inset,
values for oxidized LDL degradation at 100 µg/ml acetyl LDL
competitor are plotted as a function of the percent aggregation of each
oxidized preparation (R
=
0.88).
Khoo and colleagues (55) reported
that aggregated native LDL was internalized by LDL receptor-facilitated
phagocytosis and that this was inhibited by cytochalasin D. To
determine if an LDL receptor-independent but otherwise analogous
phagocytic pathway was involved in the uptake of aggregates of oxidized
LDL, we assessed the effect of cytochalasin D (an inhibitor of
phagocytosis) on the degradation of oxidized LDL. Ten µg/ml acetyl I-LDL, very extensively oxidized
I-LDL (23%
aggregates, electrophoretic mobility 1.04),
I-LDL
oxidized at high LDL concentration (36% aggregates, electrophoretic
mobility 0.89), or
I-LDL vortexed for 20 s (absorbance
increase at 680 nm of 1 mg/ml solution = 0.9) were incubated
with macrophages for 5 h in the absence or in the presence of 0.04
µg/ml cytochalasin D. Cytochalasin D (0.04 µg/ml) inhibited the
degradation of vortex aggregated LDL by 79 ± 3%, but did not
affect the degradation of acetyl LDL or of either oxidized LDL
preparation. It should be noted that the turbidity (reflecting mean
particle diameter) of aggregated LDL was substantially greater than
that of either oxidized LDL preparation. This suggests that although
oxidized LDL contains some aggregates, these are smaller than those in
vortex-aggregated LDL and are not handled by the same phagocytic
mechanism involved in uptake of vortex-aggregated LDL.
The preceding
results indicate that extent of aggregation of oxidized LDL correlates
with the proportion of its uptake by mouse peritoneal macrophages that
is resistant to competition by acetyl LDL, but they do not prove that
aggregation per se is the cause of this. We hypothesized that
aggregates of modified LDL would have a higher apparent affinity for
the scavenger receptor than monomeric lipoproteins because of their
potential to interact with numerous receptor molecules, and that this
might explain why acetyl LDL was unable to compete effectively for
aggregates of oxidized LDL. Inspection of the curves shown in Fig. 2suggests that if this hypothesis is true, then the
apparent affinity of aggregated oxidized LDL must be orders of
magnitude greater than that of monomeric oxidized LDL. To test this, we
generated I-LDL preparations with varying degrees of
oxidation, separated these by brief ultracentrifugation into aggregated
and nonaggregated fractions, and tested the ability of acetyl LDL to
compete for the uptake of each fraction. Results in Fig. 5show
that extensively oxidized LDL was relatively resistant to competition
by acetyl LDL, but that this was the same for aggregated and
nonaggregated fractions, and hence could not be attributed to
aggregation alone. The notion that aggregates of modified LDL would
have a higher apparent affinity for the scavenger receptor than
monomeric lipoproteins because of their potential to interact with
numerous receptor molecules was also tested by comparing the ability of
vortex-aggregated acetyl LDL to compete for the degradation of oxidized
LDL. Mouse peritoneal macrophages were incubated with 5 µg/ml
``standard'' oxidized
I-LDL (electrophoretic
mobility 0.83, 21% aggregates by electrophoresis), extensively oxidized
I-LDL (mobility 1.06, 64% aggregates), vortexed
``standard'' oxidized
I-LDL (mobility 0.83, 36%
aggregates),
I-LDL oxidized at high LDL concentration
(mobility 0.75, 48% aggregates), or acetyl
I-LDL
(mobility 1.06) in the presence or absence of up to 150 µg/ml
unlabeled acetyl LDL or acetyl LDL that had been aggregated by brief
vortexing, which increased turbidity (absorbance at 680 nm) of a 1
mg/ml solution by 0.36. The competition profiles for the various
oxidized LDL preparations were very similar to those shown in Fig. 2, and were essentially identical for untreated and
vortexed acetyl LDL. A final test of the hypothesis that aggregation
affects the apparent affinity of oxidized LDL for the scavenger
receptor type I was carried out on CHO cells stably transfected with an
expression vector containing human scavenger receptor type I cDNA.
Results in Fig. 6show that acetyl LDL was unable to compete
fully for oxidized LDL degradation in transfected cells, and that there
was no difference between its ability to compete for aggregated and
non-aggregated oxidized LDL. Taken together, these findings lead one to
reject the postulate that an aggregation-related increase in affinity
of oxidized LDL for the scavenger receptor type I is responsible for
the failure of acetyl LDL to compete for its uptake, and supports the
hypothesis that there is a second pathway.
Figure 5:
Aggregation accounts for only part of the
uptake of oxidized LDL by alternate (non-scavenger receptor type I/II)
pathways in mouse peritoneal macrophages. Three preparations of
oxidized I-LDL that differed in their degree of oxidation
were separated by ultracentrifugation into aggregated (solid
symbols) and nonaggregated (open symbols) fractions.
Mouse peritoneal macrophages were incubated with 5 µg/ml labeled
oxidized LDL together with the indicated concentration of unlabeled
acetyl LDL. After 5 h, the amount of cell-associated LDL (squares) and degraded LDL (circles) was measured. A, oxidized LDL with electrophoretic mobility 0.6 relative to
albumin (nonaggregated oxidized LDL); B, oxidized LDL with
mobility 0.94; C, oxidized LDL with mobility 1.04. Respective
100% values for degradation and cell association of nonaggregated
oxidized LDL in panel A were: 2.9 and 3.3 µg/mg, and of
aggregated oxidized LDL, 1.3 and 4.3 µg/mg. Values for degradation
and cell association of nonaggregated oxidized LDL in panel B were: 7.3 and 9.7 µg/mg, and of aggregated oxidized LDL, 3.8
and 13.1 µg/mg. Values for degradation and cell association of
nonaggregated oxidized LDL in panel C were: 7.3 and 23.3
µg/mg, and of aggregated oxidized LDL, 6.5 and 30.5
µg/mg.
Figure 6:
Aggregation does not account for
incomplete competition for uptake and degradation of oxidized LDL by
acetyl LDL in CHO cells stably transfected with human scavenger
receptor type I. CHO cells were transfected with plasmid pRC-CMV
containing human scavenger receptor cDNA, and G418-resistant clones
were selected for ability to internalize diI-labeled acetyl LDL. Panel A, varying concentrations of acetyl I-LDL
were incubated for 5 h with transfected cells (solid symbols)
or control CHO cells (open symbols), and then degradation
products in the media were measured. Panel B, 5 µg/ml
acetyl
I-LDL (
), nonaggregated oxidized
I-LDL (
), or aggregated oxidized
I-LDL
(
) were incubated with the indicated concentration of unlabeled
acetyl LDL for 5 h, and then LDL degradation products were
assayed.
CD36 is a cell surface
adhesion molecule expressed in human macrophages and endothelial cells
that has been proposed as a potential ``receptor'' for
oxidized LDL(42) . Unfortunately, neither an antibody to the
murine analog of CD36 nor a CD36 knockout mouse is presently available,
and so it was not possible to test directly if CD36 accounted for the
component of oxidized LDL uptake that could not be competed for by
acetyl LDL in mouse macrophages. To address this in human macrophages,
we first carried out cross-competition experiments between oxidized LDL
and acetyl LDL in human monocyte-derived macrophages and in THP-1 cells
that had been induced to differentiate to a macrophage-like phenotype
with phorbol ester. It was found that, as with mouse peritoneal
macrophages, acetyl LDL competed for part but not all of the
degradation of oxidized LDL (Fig. 7). However, in contrast to
results with murine cells, in human macrophages oxidized LDL was a
rather poor competitor for the degradation of acetyl LDL. It was not
ascertained if this was due to a species difference in affinity of the
scavenger receptor type I/II for oxidized LDL or to other factors such
as a second receptor specific for acetyl LDL(37) . In human
monocyte-derived macrophages, antibody to CD36 inhibited the uptake and
degradation of ``standard'' oxidized LDL by about 25% (Fig. 8). The same concentration of antibody inhibited the
binding of I-thrombospondin, a putative ligand of CD36,
to THP-1 cells by more than 50% (not shown). This is consistent with
the notion that part of the uptake of oxidized LDL might be mediated by
CD36. However, the anti-CD36 antibody inhibited the uptake and
degradation of acetyl LDL to the same extent as very extensively and/or
aggregated oxidized LDL. Therefore, it is difficult to implicate CD36
as being responsible for the failure of acetyl LDL to compete for the
uptake of these forms of oxidized LDL.
Figure 7:
Incomplete cross-competition between
oxidized LDL and acetyl LDL in human macrophages. Cultured human THP-1
macrophages (panels A and C) or human
monocyte-derived macrophages (panels B and D) were
incubated for 5 h with 5 µg/ml acetyl I-LDL (panels A and B) or 5 µg/ml oxidized
I-LDL (panels C and D) and the
indicated concentration of unlabeled acetyl LDL (
), extensively
oxidized LDL (
), or native LDL (
). Degradation products
in the medium were then measured.
Figure 8:
Inhibition of uptake of modified LDL by
antibody to CD36. Human monocyte-derived macrophages were incubated for
5 h with 5 µg/ml ``standard'' oxidized I-LDL (panel A), extensively oxidized
I-LDL (panel B),
I-LDL oxidized at
high concentration (panel C), or acetyl
I-LDL (panel D) together with the indicated concentration of
monoclonal antibody to human CD36. Cell-associated radioactivity
(
) and LDL degradation products (
) were then measured. At 4
µg/ml, this antibody inhibited the binding of
I-thrombospondin to THP-1 cells by more than
50%.
In the present studies, we have shown that the apparent
affinity of oxidized LDL as a competitor for acetyl LDL uptake in
macrophages is dependent on the extent of oxidation of LDL, even though
all of the oxidized LDL preparations had electrophoretic mobility at
least 3-fold greater that of native plasma LDL and were capable of
interacting with macrophage scavenger receptors. This effect of the
degree of oxidation has not generally been appreciated, as many
investigators simply assess their oxidized LDL preparations to verify
that oxidation has occurred, and do not determine precisely how
extensive the modification is. With the standard protocol for
generating copper-oxidized LDL used in this report (200 µg/ml LDL
incubated at 37 °C with 5 µM Cu in
PBS), incubation periods between 18 and 24 h have generally been used
for generating oxidized LDL. The rate of LDL oxidation is directly
correlated with copper concentration, and inversely correlated with LDL
concentration, LDL antioxidant content, and the concentration of metal
ion-binding substances, and some of these factors could lead to
variability between laboratories, or even between LDL preparations in
the same laboratory. In the present study, we found a large difference
in the apparent uptake pathways between preparations oxidized by the
standard protocol for 18 h compared to those oxidized for 30 h. Hence,
it is perhaps not surprising that some groups found oxidized LDL to
compete for 80% or more of acetyl LDL degradation in peritoneal
macrophages(36, 38) , while others reported only about
40% competition(34, 37, 41) .
The difference in uptake between ``standard'' oxidized LDL (electrophoretic mobility about 0.85 relative to albumin) and very extensively oxidized LDL (electrophoretic mobility greater than 1.0) could reflect increased affinity of binding of monomeric very extensively oxidized LDL to scavenger receptors. Alternatively, it could be due to aggregates in preparations of very extensively oxidized LDL causing an apparent increase in affinity for the receptor because aggregated LDL particles could interact with a greater number of receptor molecules but the present studies provide no support for this possibility. Ottnad and co-workers (56) also found a correlation between the extent of LDL oxidation and its ability to compete for the binding of acetyl LDL to liposome-reconstituted hepatic scavenger receptors, although in that report the amount of oxidation required for displacement of acetyl LDL from reconstituted receptors was much less than that required in the present study for competition for acetyl LDL degradation in cultured macrophages.
A more important
issue relates to the incomplete competition by acetyl LDL for oxidized
LDL uptake and degradation in macrophages. Some investigators reported
that acetyl LDL competed for only about 40% of oxidized LDL uptake and
interpreted this as evidence for additional receptor(s) for oxidized
LDL(36, 37, 38, 42) . However,
others have found essentially complete competition by acetyl LDL (23, 41) or acetoacetylated LDL(34) . In the
present report, we show that these differences can be explained at
least in part by heterogeneity of oxidized LDL, in that very extensive
oxidation and/or aggregation of oxidized LDL correlated with reduced
ability of acetyl LDL to compete for its uptake. One possible
explanation for the inability of acetyl LDL to compete for aggregates
of oxidized LDL is that such aggregates might interact with many
identical receptor molecules, thereby increasing the apparent affinity.
However, we found no difference in the ability of acetyl LDL to compete
for the uptake and degradation of the aggregated compared to
nonaggregated fractions of oxidized I-LDL in mouse
peritoneal macrophages or CHO cells transfected with human scavenger
receptors, and aggregation of acetyl LDL by vortexing did not augment
its ability to compete for the degradation of oxidized LDL. Therefore,
a second pathway is apparently required to account for the uptake of
oxidized LDL, particularly of very extensively oxidized preparations.
Khoo and colleagues characterized the uptake by macrophages of LDL that had been aggregated by vortexing(55) , and found that the uptake involved a phagocytic mechanism that was dependent on LDL receptor binding. LDL receptor binding cannot be invoked in the present experiments because oxidation of LDL is associated with derivatization of lysine residues and fragmentation of apo B, and LDL receptor binding is abolished even after relatively modest degrees of oxidation, corresponding to electrophoretic mobility about 0.5 relative to albumin (23) . None of the oxidized LDL preparations used in the present work would have been capable of interacting with the LDL receptor, but it was possible that LDL receptor-independent phagocytosis was involved. However, cytochalasin D (an inhibitor of phagocytosis) blocked the degradation of vortexed native LDL but not that of oxidized LDL or acetyl LDL.
Reference has been made above to reports that CD36 can interact with oxidized LDL and mediate its internalization(42, 43, 44) . However, we found that although antibody to CD36 resulted in a slight inhibition of the uptake of oxidized LDL in human monocyte-derived macrophages, it affected the uptake of nonaggregated and aggregated forms of oxidized LDL and acetyl LDL to the same extent, and hence CD36 did not appear to explain the observed differences in uptake pathways for these modified LDLs. Acton and co-workers (57) cloned a new scavenger receptor termed SR-BI that is 30% homologous to CD36 and binds oxidized LDL. This receptor also binds acetyl LDL, and hence is unlikely to explain the acetyl LDL-independent component of oxidized LDL uptake that is the focus of the present work. Ottnad and co-workers (58) have described a 94-97 kDa plasma membrane protein in macrophages that binds to oxidized LDL on ligand blots and have proposed that this may represent a receptor for oxidized LDL. As well, phosphatidylcholine/cholesterol vesicles containing small amounts of acidic phospholipids are internalized and metabolized by macrophages via a pathway that is completely inhibitable by oxidized LDL but only partly by acetyl LDL (59) , and this pathway does not involve scavenger receptors type I or II(60) . Once the receptors involved in these pathways have been fully characterized, it should be possible to determine if they account for the scavenger receptor-independent component of the uptake of oxidized LDL by macrophages. In the meantime, the present results indicate that inferences based on competition studies between oxidized LDL and other scavenger receptor ligands need to be interpreted cautiously because the apparent mechanism of uptake of oxidized LDL depends on the extent of LDL oxidation.