© 1997 American Heart Association, Inc.
Articles |
Uptake by J774 Macrophages of Very-Low-Density Lipoproteins Isolated From ApoE-Deficient Mice Is Mediated by a Distinct Receptor and Stimulated by Lipoprotein Lipase
From the TNO–Prevention and Health, Gaubius Laboratory (W.L.H., B.J.M. van V., F. van der S.-de B., L.C. van V., L.M.H), and the MGC–Department of Human Genetics, Leiden University (M.H.H.), Leiden, The Netherlands.
Correspondence to Dr L.M. Havekes, TNO–Prevention and Health, Gaubius Laboratory, Zernikedreef 9, 2333 CK Leiden, PO Box 2215, 2301 CE Leiden, The Netherlands. E-mail lm.havekes{at}pg.tno.nl.
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Abstract |
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Abstract Apolipoprotein (apo) E–deficient mice display marked accumulation in the plasma of VLDL deficient in both apoE and apoB100 but containing apoB48, apoA-I, apoCs, and apoA-IV. Since apoE-deficient mice develop severe atherosclerotic lesions with lipid-laden macrophages, we reasoned that the uptake of lipoproteins by intimal macrophages can take place in the absence of both apoE and apoB100. To get more insight into the mechanism of foam cell formation in apoE-deficient mice, we measured the interaction of VLDL from apoE-deficient mice (apoEnull VLDL) with the murine macrophage cell line J774. Scatchard analysis revealed that apoEnull VLDL is bound to J774 cells with a Kd value comparable to that of control VLDL (8.1 versus 4.7 µg/mL) and with a Bmax value about half that of control VLDL (40 versus 70 ng/mg cell protein, respectively). ApoEnull VLDL is also taken up and degraded by J774 macrophages via a high-affinity process less efficiently than control mouse VLDL (6-fold and 50-fold less efficiently, respectively). In line with this observation, incubation of J774 cells with 50 µg/mL apoEnull VLDL for 24 hours resulted in an increase in intracellular cholesteryl ester (CE) content, although 5-fold less pronounced than after incubation with 50 µg/mL control mouse VLDL. Under the conditions applied, simultaneous addition of 5 µg/mL lipoprotein lipase (LPL) stimulated the cellular uptake and degradation of apoEnull VLDL about 10-fold and resulted in a 5-fold stimulation of the intracellular CE accumulation, from 9±2 to 46±5 µg CE per milligram cell protein. In contrast to control mouse VLDL, apoEnull VLDL could not compete with 125I-labeled LDL for binding to the LDL receptor of J774 cells. Furthermore, neither LDL nor acetylated LDL could compete with 125I-labeled apoEnull VLDL for binding to these cells, whereas control mouse VLDL, VLDL from a hypertriglyceridemic patient, and apoEnull VLDL itself were efficient competitors. Thus, VLDL from apoE-deficient mice is taken up by J774 macrophages through recognition by a distinct receptor, which could be the triglyceride-rich lipoprotein receptor. We conclude that in apoE-deficient mice, foam cell formation occurs via a receptor-mediated uptake of apoEnull VLDL, which can be stimulated by the presence of LPL.
Key Words: apoE-deficient mice • macrophages • foam cells • receptor-mediated uptake • lipoprotein lipase
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Apolipoprotein E is a major protein constituent of chylomicrons, VLDL, chylomicron and VLDL remnants, and HDL. One of the major functions of apoE in lipoprotein metabolism is that it serves as a ligand for hepatic receptors, mediating the removal of apoE-containing lipoproteins from the circulation.
By using gene targeting in murine embryonic stem cells, several groups have generated mice lacking apoE.1 2 3 These mice exhibit extreme hypercholesterolemia that is much more severe than that observed in apoE-deficient humans.4 5 6 The reason for this difference might be the fact that in mice, particles containing both apoB100 and apoB48 are produced by the liver, whereas in humans, the liver produces only particles containing apoB100.7 8 In contrast to apoB100, apoB48 does not function as a ligand for hepatic lipoprotein receptors, and therefore, apoE-deficient mice display a marked accumulation of chylomicrons and VLDL that are deficient in both apoE and apoB100 but that contain apoB48.1 Since apoE-deficient mice develop extensive atherosclerotic lesions with lipid-laden macrophages, even on a regular chow diet,9 we suggest that uptake of lipoprotein particles into macrophages present in the intima can occur in the absence of both apoB100 and apoE. Hence, additional uptake mechanisms may play a role in the process of foam cell formation in apoE-deficient mice.
To provide more insight into the formation of foam cells in mice lacking apoE, we studied the processing of apoEnull VLDL by J774 macrophages, a murine macrophage cell line that is commonly used as a model for foam cell formation and that does not produce apoE.10
We also investigated the role of LPL in binding and uptake of apoEnull VLDL by J774 cells, since LPL is synthesized by macrophages present in the intima11 and is known to enhance the binding and uptake of LDL and VLDL by different cell types, including macrophages.12 13 14 15 16 17
Our results show that VLDL deficient in both apoE and apoB100 is taken up by J774 macrophages via a receptor-mediated pathway, leading to the accumulation of CE in these cells. In addition, like normal VLDL, this apoEnull VLDL can also be taken up via an LPL-mediated process.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Cells
Murine macrophage-like J774 cells were cultured in 75-cm2 flasks in DMEM supplemented with 10% (vol/vol) FCS, 0.85 g/L NaHCO3, 4.76 g/L HEPES, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L glutamine. The cells were incubated at 37°C in an atmosphere containing 5% CO2 in air. For each experiment, cells were plated in 12- or 24-well plates. The cells were fed every 3 days and used for experiments within 7 days of plating.
Animals and Lipoproteins
Mice were allowed access to food and water ad libitum.
ApoE-deficient mice were generated as described1 and fed a
regular chow diet (SRM-A). The control mice were of a genetic
background similar to that of the apoE-deficient mice (129
SvxC57BL/6J). To obtain lipoprotein samples from control mice with a
lipid composition comparable to that of apoE-deficient mice, control
mice were fed a semisynthetic severe high-fat/cholesterol (HFC 0.5%)
diet18 for a period of 4 weeks, composed essentially
according to Nishina et al19 and purchased from Hope
Farms, Woerden, The Netherlands. This diet contains 15% cocoa butter,
1% cholesterol, 0.5% cholate, 40.5% sucrose, 10% cornstarch, 1%
corn oil, and 4.7% cellulose (all percentages are by weight) and all
the required nutrients, minerals, and vitamins. After a 4-hour fasting
period, mice were anesthetized with diethyl ether. Blood was collected
from apoE-deficient (n=11) and control (n=62) mice by orbital puncture.
The d<1.006 g/mL lipoproteins (VLDL) were isolated from the
mouse serum by density gradient ultracentrifugation. Human serum was
prepared from freshly collected blood either from healthy volunteers or
from a type IV hyperlipidemic patient. Human LDL and HTG-VLDL were
isolated from the respective sera by density gradient
ultracentrifugation as described by Redgrave et al.20 A
portion of the LDL sample was acetylated by repeated addition of acetic
anhydride as described by Basu et al.21 The conversion of
LDL into AcLDL was confirmed by agarose gel electrophoresis (100 V, 30
minutes, Paragon lipoprotein electrophoresis kit, Beckman Instruments
Inc). The protein content of the lipoprotein samples was determined
according to Lowry et al.22
Total and free cholesterol, triglyceride (without free glycerol), and phospholipid contents of the lipoproteins were measured enzymatically, using commercially available kits (Nos. 236691 and 310328 from Boehringer Mannheim GmbH, No. 337-B from Sigma Chemical Company, and No. 990-54009 from Wako Chemicals GmbH, respectively).
To determine the relative apolipoprotein composition of the
lipoproteins, 
12.5 µg of lipoprotein protein was applied on
SDS–polyacrylamide gradient gels (4% to 20%). After electrophoresis,
the protein was stained with Coomassie brilliant blue, followed by
densitometric scanning of the bands with a Hewlett Packard ScanJet
Plus.
Mouse VLDL and human LDL were radioiodinated by using the 125I-monochloride method described by Bilheimer et al.23 The specific activity ranged from 90 to 400 cpm/ng protein. After iodination, the lipoprotein samples were dialyzed extensively against PBS (pH 7.4), stabilized with 1% (wt/vol) HSA, stored at 4°C, and used within 2 weeks.
Whenever unlabeled lipoproteins were used, immediately after isolation, dialysis against PBS containing 10 µmol/L EDTA and subsequently DMEM was performed at 4°C.
LPL
LPL was partly purified from fresh bovine milk by using
heparin-Sepharose chromatography as described
previously.24
Binding and Uptake of Lipoproteins by J774 Macrophages
The J774 cells were cultured in 24-well plates as described
above. Twenty-four hours before each experiment, the cells were washed
with DMEM containing 1% (wt/vol) BSA and further incubated with DMEM
containing 5% (vol/vol) lipoprotein-deficient serum
(d<1.21 g/mL) instead of FCS. The binding and uptake
studies were subsequently conducted in medium containing no serum
components other than the respective lipoproteins and 1% (wt/vol)
BSA.
The binding of 125I-labeled lipoproteins to the cells in the absence or presence of 5 µg/mL LPL was determined after a 3-hour incubation at 4°C with different concentrations of 125I-labeled lipoprotein, either in the presence or absence of a 20-fold excess of the respective unlabeled lipoprotein. The concentrations of the labeled lipoproteins in the respective incubations are described in the text and figure legends. The receptor-mediated (specific) cell binding was calculated by subtracting the amount of labeled lipoproteins that was cell bound after incubation in the presence of the excess of unlabeled lipoprotein (nonspecific) from the amount of labeled lipoprotein that was cell bound after incubation in the absence of unlabeled lipoprotein (total cell binding). After removal of the medium, the cells were washed four times with ice-cold PBS containing 0.1% (wt/vol) BSA and subsequently once with PBS without BSA. Cells were then dissolved in 1 mL of 0.2 mol/L NaOH. Protein content was measured by the method of Lowry et al.22 In an aliquot, the radioactivity represented the amount of cell-bound lipoprotein.
The association and degradation of 125I-labeled lipoproteins in the absence or presence of 5 µg/mL LPL was determined after a 3-hour incubation at 37°C with 10 µg/mL 125I-labeled lipoprotein either in the absence or presence of 200 µg/mL of the respective unlabeled lipoprotein. At the end of the incubation period, a fraction of the medium was removed to determine the amount of lipoprotein degraded, as described previously.25 26 After removing the remaining portion of the medium, the cells were washed four times with ice-cold PBS containing 0.1% (wt/vol) BSA and subsequently with PBS without BSA. To measure the cell-associated (bound plus internalized) lipoprotein fraction, the washed cells were dissolved in 1 mL 0.2 mol/L NaOH and an aliquot of the cell lysate was counted for radioactivity.25 Another aliquot was used for protein determination according to Lowry et al.22
To identify the nature of the receptor that mediates the binding of apoEnull VLDL to J774 macrophages, competition experiments were performed in which J774 cells were incubated for 3 hours at 4°C with either 10 µg/mL 125I-labeled human LDL, 10 µg/mL 125I-labeled apoEnull VLDL, or 10 µg/mL 125I-labeled HTG-VLDL in the presence of different concentrations of the indicated unlabeled lipoprotein samples. Thereafter, cells were washed and binding at 4°C was measured as described above.
Cellular Accumulation of Lipid
J774 cells were cultured in 12-well plates as described above.
Twenty-four hours before the start of the experiment, DMEM supplemented
with 5% (vol/vol) lipoprotein-deficient serum instead of FCS was added
to the cells. At the start of the experiment, fresh DMEM containing 1%
HSA (wt/vol) and 50 µg/mL lipoprotein protein either in the presence
or absence of 5 µg/mL LPL was added in triplicate dishes of cells and
incubated for 24 hours at 37°C. Control incubations were performed
with DMEM/1% HSA without any further additions or with DMEM/1% HSA
with 5 µg/mL LPL. At the end of the incubation period, the cells were
washed four times with 1 mL of PBS containing 0.1% (wt/vol) BSA
followed by one wash with PBS alone. Intracellular lipid content was
determined as described by Havekes et al.27 Briefly, the
cells were harvested by scraping with a rubber policeman and
resuspended by three successive slow passages through a syringe needle
(G25). Samples (100 µL) were taken for measurement of protein. Lipids
were extracted from the cell suspension using methanol/chloroform (2:1,
vol/vol) as described by Bligh and Dyer28 after addition
of cholesteryl acetate (2 µg) as an internal standard. The lipids
were separated using high-performance thin layer chromatography.
Subsequently the lipid bands were quantified densitometrically on a
Shimadzu CS910 chromatograph scanner at 380 nm, and areas under the
curve were integrated by using a data processor.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Lipid and Apolipoprotein Composition of ApoEnull VLDL and Control VLDL
As expected, the cholesterol, triglyceride, and phospholipid levels are similar in VLDL isolated from control mice fed a high-fat, high-cholesterol diet (HFC 0.5%) and in VLDL from apoE-deficient mice fed a regular chow diet (Table 1
).
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In apoEnull VLDL, apoB48, apoA-IV, and apoCs are the major
protein constituents, whereas small amounts of apoA-I are also found
(Fig 1). No apoB100 or apoE could be detected in this
lipoprotein sample. In control mouse VLDL, apoE is the major
apolipoprotein, whereas equal amounts of apoB100 and apoB48 are found,
in addition to apoA-IV and apoCs.
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Binding and Uptake of Lipoproteins by J774 Macrophages
J774 macrophages were incubated with increasing concentrations of
125I-labeled apoEnull VLDL or control VLDL in
the absence or presence of a 20-fold excess of unlabeled lipoprotein
for 3 hours at 4°C to measure the total and nonspecific binding,
respectively. In Fig 2A and 2B, the upper curve
represents total binding (specific plus nonspecific) of
apoEnull VLDL and control VLDL, respectively, whereas the
lower curve represents nonspecific binding, which occurs in the
presence of excess unlabeled lipoprotein. The calculated difference
between the two curves represents specific binding (middle curve). J774
cells express a saturable binding site for both apoEnull
VLDL and control VLDL. Scatchard plot analysis, shown in the insets of
the graphs, reveals that the dissociation constants
(Kd) are 8.1 and 4.7 µg/mL for
apoEnull VLDL and control VLDL, respectively; the
Bmax of apoEnull VLDL and control VLDL is 40
and 70 ng/mg cell protein, respectively.
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As presented in Fig 3A, at 10 µg labeled lipoprotein
per milliliter, the high-affinity (receptor-mediated) binding of
125I-labeled apoEnull VLDL by J774 macrophages
is low compared with 125I-labeled control VLDL (13±6
versus 57±2 ng/mg cell protein, respectively). Addition of 5 µg/mL
LPL resulted in a 20-fold increase of the binding of control VLDL to
J774 macrophages, whereas the binding of apoEnull VLDL was
stimulated about 100-fold on addition of LPL (compare Fig 3A with 3B
and note the difference in scale).
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ApoEnull VLDL was also associated (bound plus internalized)
and degraded by J774 cells via a high-affinity process but to a much
lesser extent than control VLDL (55±5 and 15±10 versus 315±19 and
642±22 ng/mg of cell protein, respectively; Fig 3C
and 3E). The
association and degradation of control VLDL by J774 cells were
stimulated 2-fold and 1.5-fold, respectively, on addition of 5
µg/mL LPL. The association and degradation of apoEnull
VLDL were enhanced by a factor of 8 and 16, respectively, in the
presence of 5 µg/mL LPL (compare Fig 3C with 3D and 3E with 3F,
respectively).
Our data indicate that 125I-labeled apoEnull
VLDL is bound and internalized by J774 cells via a specific saturable
receptor, since its binding, association, and degradation are reduced
on addition of an excess amount of unlabeled apoEnull VLDL.
To identify the nature of the receptor involved in the binding and
uptake of apoEnull VLDL, competition experiments were
performed. On addition of 20 µg/mL unlabeled LDL, the binding of
125I-labeled human LDL by J774 cells was reduced to about
60% of the control binding (Fig 4). Unlabeled control
VLDL was much more efficient in competing with 125I-labeled
LDL (reduction up to 30% of the control binding). This high efficiency
of control VLDL in competing for the binding of LDL can be explained by
the fact that control mouse VLDL is relatively rich in apoE, whereas
human LDL consists of apoB100 only. ApoEnull VLDL was not
able to compete with 125I-labeled LDL for binding to the
J774 cells. As shown in Fig 5, the binding of
125I-labeled apoEnull VLDL by J774 cells was
inhibited to a similar extent (60% of control value) on addition of
either unlabeled apoEnull VLDL, control VLDL, or human
HTG-VLDL. On the contrary, addition of unlabeled LDL or AcLDL did not
significantly reduce the binding of
125I-apoEnull VLDL. The binding of
125I-labeled HTG-VLDL to the cells was inhibited to 20%
of control value by both unlabeled HTG-VLDL and control VLDL and to
55% of control value by LDL. ApoEnull VLDL and AcLDL were
not able to compete with 125I-labeled HTG-VLDL for binding
to J774 macrophages. From these experiments we conclude that the
binding of apoEnull VLDL does not occur via the LDL
receptor or the scavenger receptor but that a receptor also recognizing
control mouse VLDL and HTG-VLDL is involved in the binding of
apoEnull VLDL to J774 macrophages.
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),
apoEnull VLDL (
), and control VLDL (
). The binding
is expressed as a percentage of the value in the absence of competitor
and is determined as described in "Methods." Values represent the
mean±SD of four measurements.
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), or HTG-VLDL (
).
The binding is expressed as a percentage of the value in the absence of
competitor and is determined as described in "Methods." Values
represent the mean±SD of four measurements.
Accumulation of CE in J774 Macrophages After Incubation With
Different Lipoproteins
As apoE-deficient mice develop atherosclerotic plaques containing
lipid-laden macrophages (foam cells), we wondered whether
apoEnull VLDL, which is deficient in both apoE and apoB100,
can cause accumulation of CE in J774 macrophages. The data presented in
Table 2 demonstrate that incubation of J774 cells with
apoEnull VLDL resulted in a threefold increase in cellular
CE content. Simultaneous incubation with LPL stimulated the cellular CE
content by an additional factor of 5, reaching similar levels as
obtained after incubation with control VLDL. However, simultaneous
addition of LPL did not stimulate the cellular CE level in J774 cells
after incubation with control VLDL.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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ApoE-deficient mice have been shown to display a marked accumulation in the plasma of chylomicron and VLDL remnants, which are deficient in both apoE and apoB100. ApoE and apoB100 are also commonly assumed to be responsible for the receptor-mediated uptake of lipoproteins by macrophages. Thus, since apoE-deficient mice develop extensive atherosclerotic lesions with lipid-laden macrophages, uptake of lipoproteins by macrophages can take place in the absence of both apoB100 and apoE. In the present paper we studied the mechanisms by which apoEnull VLDL can be taken up by macrophages by using the murine macrophage cell line J774, which does not produce apoE itself.10
We found that apoEnull VLDL is bound by J774 macrophages
via a high-affinity, saturable receptor with a dissociation constant
(Kd) of the same order of magnitude as that of
control VLDL (Fig 2). Furthermore, uptake of apoEnull VLDL
resulted in the accumulation of CE in these cells. The LDL receptor was
not expected to be a candidate for the uptake of apoEnull
VLDL, since apoE and apoB100, which are both absent on the surface of
this VLDL, are known to be the only ligands for this receptor. We found
that the scavenger receptor was also not involved in the uptake by J774
macrophages of apoEnull VLDL. However, since the scavenger
receptor is involved in the uptake of oxidized lipoproteins by
macrophages29 and lipoprotein oxidation occurs in the
intima of apoE-deficient mice,30 31 32 it is plausible that
in vivo, the scavenger receptor does indeed contribute to foam cell
formation and subsequent development of atherosclerosis in
apoE-deficient mice.
Gianturco et al33 34 and Ramprasad et al35 36
described a distinct receptor that is able to mediate the uptake of
HTG-VLDL and that has been denominated as the TGRLP receptor. They
reported that this receptor plays a major role in the uptake of
HTG-VLDL by human monocyte-macrophages33 35 36 and murine
macrophages.34 Recently, Gianturco et al37
reported that neither apoB100 nor apoE is necessary to mediate the
binding of HTG-VLDL to this receptor and that apoB48 is sufficient for
this purpose. The fact that apoB48 and not apoE or apoB100 is present
on the surface of apoEnull VLDL supports the hypothesis
that the binding and uptake of apoEnull VLDL by macrophages
occurs via this TGRLP receptor. This hypothesis is strongly sustained
by our observation that HTG-VLDL does indeed compete with VLDL from
apoE-deficient mice for binding to the J774 macrophages (Fig 5). The
observation that an excess concentration of apoEnull VLDL
does not compete for the binding of 125I-labeled HTG-VLDL,
is not in disaccord with our hypothesis, since HTG-VLDL also binds to
the cells via apoB100 and apoE, as shown by the finding that both
control mouse VLDL and LDL can compete for the binding of
125I-labeled HTG-VLDL (Fig 6). Obviously,
this part of the HTG-VLDL binding cannot be competed with by
apoEnull VLDL.
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It has previously been shown by several groups that LPL stimulates the
binding and uptake of LDL and VLDL by different cell types, including
macrophages, via bridging between the lipoprotein and the heparan
sulfate proteoglycans present on the plasma membrane.12 13 14 15 16 17
In the present paper we present data that the binding and uptake of
apoEnull VLDL by J774 macrophages is also stimulated on
addition of LPL. The finding that the binding of VLDL is stimulated by
LPL to a greater extent than both the association and the degradation
(Fig 3) suggests that a large part of the LPL-mediated VLDL binding at
37°C is reversible. This would mean that not all the VLDL bound via
LPL to the J774 cells is taken up and subsequently degraded. Another
explanation could be that under the conditions applied (J774 cells
incubated 3 hours at 37°C), the LPL-bound VLDL is not internalized
and/or degraded as rapidly as VLDL bound to the receptor directly. At
present we cannot discriminate between the two possibilities. The
increased LPL-mediated uptake of apoEnull VLDL resulted in
an increased accumulation of CE in these cells (Table 2). In vivo, LPL
is synthesized by macrophages, positively correlated with the amount of
intracellular cholesterol.11 Furthermore, in
atherosclerotic lesions, high amounts of LPL protein are formed mainly
in macrophage-rich intimal regions.38 Thus, in
apoE-deficient mice, the LPL-mediated uptake of VLDL by macrophages may
play an important role in the development of atherosclerosis.
The role of apoE in the development of atherosclerotic lesions may be dual. On one hand, the apoE-mediated uptake of lipoproteins by cells of the arterial wall will lead to lipid accumulation and foam cell formation. On the other hand, apoE plays a role in the cholesterol efflux from cholesterol-laden macrophages to extracellular cholesterol acceptors.39 Thus, the absence in apoE-deficient mice of apoE-mediated cholesterol efflux may also contribute to the marked atherosclerosis observed in these mice. This is sustained by the finding of Bellosta et al40 that macrophage-specific expression of human apoE is able to prevent or delay the development of atherosclerotic lesions in apoE-deficient mice, even in the presence of high levels of atherogenic lipoproteins.
In summary, in addition to the absence of an apoE-mediated cholesterol efflux from macrophages in apoE-deficient mice, we present evidence that there may be at least two additional processes that contribute to the development of atherosclerosis in apoE-deficient mice. First, VLDL is taken up by macrophages via a specific saturable receptor, possibly the TGRLP receptor, which leads to the accumulation of CE in these cells. Second, this accumulation of CE by macrophages in the intima may be enhanced by production of LPL by macrophages in the intima.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study is financially supported by the Netherlands Heart Foundation (project No. 92.337). Dr M.H. Hofker is awarded a Dr E. Dekker Established Investigatorship of the Netherlands Heart Foundation (D95.022). We would like to thank Hans van der Boom for excellent technical assistance.
Received February 26, 1996; accepted July 10, 1996.
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