* Departments of Pathology,
Pharmacology, and
Medicine (Neurology), Duke University Medical Center, Durham, North Carolina
Correspondence: Daniel Laskowitz, M.D., Department of Medicine (Neurology), Box 2900, Duke University Medical Center, Durham, NC 27710.
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Key Words: apolipoprotein E • receptor-associated protein • macrophage • lipoprotein receptor-related protein • signal transduction
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ApoE has two distinct functional domains, a 10-kDa carboxyl terminus
and a 22-kDa amino terminus [12
]. The carboxyl terminus
has a high affinity for lipid and mediates the role of ApoE in
cholesterol transport. The amino terminus is composed of four
antiparallel 
helices. The motif includes the receptor-binding
region of ApoE, which is located between residues 134 and 150
[13
14
]. This region is highly enriched in basic
residues, and it is responsible for ligand binding with high affinity
to the low-density lipoprotein (LDL) superfamily of receptors,
including very-low-density lipoprotein (VLDL), lipoprotein
receptor-related protein (LRP), apolipoprotein E receptor 2
(ER)-2, and the renal glycoprotein GP330/megalin
[15
16
17
]. LRP is present on macrophages and other
macrophage-derived cells such as microglia [18
].
Depending on the ligand, binding to this receptor may result in the
activation of a pertussis toxin-coupled G protein and induction of a
typical signaling cascade [19
20
21
]. Binding of
lactoferrin, lipoprotein lipase, or Pseudomonas exotoxin A
to LRP activates this signaling cascade; however, binding of
2-macroglobulin, LDL, or receptor-associated protein
(RAP) to LRP fails to trigger signal transduction
[19
20
21
22
]. Although ApoE binds to LRP [16
17
18
23
24
], its binding is a complex phenomenon probably requiring the
participation of heparan sulfate proteoglycans [25
].
However, the ability of ApoE to regulate signal transduction has not
been studied in macrophages.
In this study, we demonstrate that ApoE initiates a receptor-mediated signaling cascade in murine peritoneal macrophages. This signal cascade is associated with internal Ca2+ mobilization and turnover of inositol triphosphate (IP3), consistent with a classical second-messenger pathway. Moreover, we determined that the receptor-binding region of ApoE was sufficient for signal transduction, suggesting that initiation of signaling is receptor-mediated and independent of lipid binding. Our results also implicate LRP as a potential receptor candidate for ApoE.
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Harvesting of murine peritoneal macrophages
All experiments involving animals were approved by the Duke
Institutional Animal Care and Use Committee. Pathogen-free female
C57BL/6 mice and ApoE-deficient mice previously backcrossed 10 times to
the C57BL/6 strain were obtained from the Jackson Laboratory (Bar
Harbor, ME). Peritoneal macrophages were obtained as previously
described [22
]. Thioglycollate-elicited peritoneal
macrophages were harvested by peritoneal lavage using 10 mL of ice-cold
Hanks’ balanced salt solution containing 10 mM HEPES and 3.5 mM
NaHCO3 (HHBSS), pH 7.4. The macrophages were pelleted by
centrifugation at 4°C at 
800 g for 10 min and
resuspended in RPMI 1640 medium supplemented with 25 mM HEPES, 12.5
U/mL of penicillin, 6.5 mg/mL of streptomycin, and 5% fetal bovine
serum. Cell viability was determined by the trypan blue exclusion
method and was consistently >95%.
Receptor-binding studies
Macrophages were plated in 48-well cell culture plates at
2.5 x 105 cells per well and incubated for 3 h
at 37°C in a humidified, 5% CO2 incubator. The plates
were then cooled to 4°C, and unbound cells were removed by three
consecutive rinses with ice-cold Hanks’ balanced salt solution
containing 20 mM HEPES and 5% BSA, pH 7.4 (binding buffer). To
quantify direct binding of the 125I-labeled ApoE-mimetic
peptide, various amounts of radiolabeled peptide were added to each
well in the presence or absence of a 200-fold molar excess of unlabeled
peptide. Specific binding to cells was determined by subtracting the
amount of 125I-labeled ApoE-mimetic peptide bound in the
presence of excess unlabeled peptide (nonspecific binding) from the
amount of 125I-labeled ApoE-mimetic peptide bound in the
absence of excess unlabeled peptide (total binding). For competition
studies, 50 nM radiolabeled peptide was added to each well in the
presence or absence of various amounts (31.25 nM–4 µM) of unlabeled
ApoE or RAP. Cells were then incubated at 4°C for 12–16 h. Unbound
ligand was removed from the wells, and the cell monolayer was rinsed
three times with ice-cold binding buffer. Cells were then solubilized
with 1 M NaOH and 0.5% SDS at room temperature for >5 h before the
contents of each well were added to polystyrene tubes and counted in an
LKB-Wallac, CliniGamma 1272 
-counter (LKB-Wallac, Turku,
Finland).
Measurement of intracellular calcium levels
Intracellular Ca2+ in Fura-2/AM-treated single cells
was quantified using digital imaging microscopy as described previously
[20
21 23
]. Macrophages were plated on glass
coverslips placed in 35-mm Petri dishes at a density of 1.5 x
105 cells/cm2 and allowed to adhere for 2 h in a humidified 5% CO2 incubator at 37°C.
The nonadherent cells were aspirated, and the monolayers were washed
twice with HHBSS. Fura-2/AM (4 µM) was incubated with the cells for
30 min in the dark at room temperature, and
[Ca2+]i was subsequently measured using
digital-imaging microscopy as previously described
[19
20
21
22
]. After obtaining baseline measurements for 5
min, a ligand [ApoE (100 pM), ApoE-mimetic peptide (100 nM), or
scrambled peptide (100 nM)] was added, and multiple
[Ca2+]i measurements were taken. To determine
whether signaling resulted from ligation of the ligand to LRP, cells
were preincubated with a 1,000-fold molar excess of RAP or 10 mM
NiCl2, both of which inhibit ligand binding to LRP
[20
], for 10 min before stimulation with ApoE or
peptide. In experiments in which the involvement of a G-protein was
assessed, monolayers were incubated with 1 µg/mL of pertussis toxin
(preactivated with 40 mM dithiothreitol at 30°C for 20 min) for
12 h at 37°C before Ca2+ measurements. To deplete
intracellular Ca2+, the cells were incubated with 1 mM
thapsigargen (TG) for 10 min before stimulation by the ligand.
Measurement of inositol phosphates
The formation of IP3 in
myo-[2-3H]inositol-labeled macrophages under various
experimental conditions was quantified as previously described
[26
]. Macrophages were plated in six-well plates
(4x106 cells/well) and allowed to adhere at 37°C for
2 h in a humidified 5% CO2 incubator. The
medium was aspirated from the monolayers and RPMI 1640 medium
containing 0.25% BSA and myo-[2-3H]inositol (specific
activity, 10–20 Ci/mmol) was added to each well. The cells were
incubated at 37°C for an additional 16–18 h. The monolayers were
then rinsed three times with 25 mM HHBSS containing 1 mM
CaCl2, 1 mM MgCl2, and 10 mM LiCl, pH 7.4. A
volume of 0.5 mL of this solution was added to each well, and the cells
were preincubated for 3 min at 37°C before the addition of the
ligand. The reaction was stopped by aspirating the medium containing
the ligand and adding 6.25% ice-cold perchloric acid. The cells were
scraped out of the wells, transferred to tubes containing 1 mL of
octylamine/Freon [1:1 (v/v)] and 5 mM EDTA, and were centrifuged at
5,600 g for 20 min at 4°C. The upper-phase solution was
applied to a 1-mL Dowex resin column (AG1-X8 formate; BioRad
Laboratories, Richmond, CA) and eluted sequentially in a batch process
with H2O and 50, 200, 400, 800, and 1,200 mM ammonium
formate containing 0.1 M formic acid [26
]. Radioactivity
was determined by placing aliquots in a liquid scintillation counter to
determine radioactivity. To evaluate the pertussis-toxin sensitivity of
the G-protein coupled to receptor activation and phosphatidyl inositol
4,5-bisphosphate (PIP2) hydrolysis, cells were incubated
with 1 µg/mL of pertussis toxin before addition of the ligand.
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 View larger version (13K): [in a new window] |
Figure 1. [Ca2+]i measurements in macrophages exposed
to ApoE. (A) Measurement of [Ca2+]i in a
single macrophage on stimulation with ApoE (100 pM). The graph shown is
a single cell tracing representative of five individual experiments
using 20–30 cells each. Approximately 70–80% of the macrophage
responded with changes in [Ca2+]i on
stimulation with ApoE. Arrows (A and C) indicate the times of ApoE
addition. (B) Effect of ApoE concentration on percent change in
[Ca2+]i over baseline. The changes in
[Ca2+]i in individual cells were measured
before and after exposure to various concentrations of ApoE. Data
represent the means ± SE of the
[Ca2+]i change for all cells from two
independent experiments in which 25–30 cells were analyzed. (C)
Measurement of [Ca2+]i in a single macrophage
pretreated with TG (left arrow) (1 µM) for 10 min then stimulated
with ApoE. The graph shown is a single cell tracing representative of 5
individual experiments using 20–30 cells each. The right arrow
indicates the time of ApoE addition. For all panels, details for
[Ca2+]i measurement are described in
Materials and Methods.
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We next sought to determine whether the native ApoE secreted by macrophages affected the increase in [Ca2+]i that was induced by the addition of exogenous ApoE. To address this possibility, these experiments were repeated using macrophages prepared from ApoE-deficient mice. The Ca2+ responses after stimulation with ApoE were identical between wild-type macrophages and macrophages from ApoE-deficient mice (data not shown), thus displaying that native ApoE did not affect exogenous signaling.
Effect of ApoE on inositol phosphate formation
In many cell types, the binding of ligands to plasma
membrane receptors activates hydrolysis of PIP2 by
membrane-bound phospholipase C, leading to the formation of
IP3. IP3 then causes the release of
Ca2+ from the endoplasmic reticulum by binding to its
cognate receptor, which is also a Ca2+ channel. To
determine whether the ApoE-induced [Ca2+]i
response was occurring through this pathway, myo-[2-3H]
inositol-labeled macrophages were exposed to ApoE, and IP3
formation was measured. ApoE binding resulted in a 1.5- to 2-fold
increase in IP3 levels (Fig. 2A
). The magnitude of the increase in IP3 formation was
dependent on the concentration of the ApoE dose (Fig. 2B)
. These data
suggest that the [Ca2+]i increases caused by
ApoE result from increased IP3 formation.
 View larger version (13K): [in a new window] |
Figure 2. IP3 formation in macrophages exposed to apoE. (A) The
effect of ApoE on IP3 synthesis in macrophages with and
without the addition of pertussis toxin.
Myo-[2-3H]inositol-labeled cells were stimulated with
ApoE (100 pM) in the absence (•) or presence ( ) of pertussis
toxin. (B) Effect of ApoE concentration on the maximal change in
IP3 formation over baseline. Cells were
stimulated with various concentrations of ApoE for 60 s, and
IP3 formation was measured. For both panels,
data represent the mean ± se of all the cells from two
individual experiments performed in duplicate. Details for the
measurement of IP3 formation are described in Materials and
Methods.
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 View larger version (15K): [in a new window] |
Figure 3. [Ca2+]i measurements in macrophages exposed
to ApoE in the presence of pertussis toxin. Macrophages were
preincubated for 16 h with pertussis toxin (1 µg/mL) () or
buffer (•) and then were exposed to ApoE (100 pM) at the time
indicated by the arrow. Data shown are single cell tracings
representative of two independent experiments each in which 25–30
cells were analyzed. The details for [Ca2+]i
measurement are described in Materials and Methods.
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 View larger version (18K): [in a new window] |
Figure 4. [Ca2+]i measurements in macrophages exposed
to ApoE in the presence of RAP and Ni2+. Macrophages were
preincubated with buffer (•), RAP (0.1 µM) ( ), or
NiCl2 (10 mM) ( ) and then exposed to ApoE (100 pM) at
the time indicated by the arrow. The data shown are single cell
tracings representative of two independent experiments each, in which
25–30 cells were analyzed. The details for
[Ca2+]i measurement are described in
Materials and Methods.
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60–70% of cells examined. As with the ApoE responses,
peptide-induced increases in macrophage
[Ca2+]i were heterogeneous and asynchronous.
These results demonstrate that both intact ApoE and a peptide derived
from the ApoE receptor-binding region induce an increase in
[Ca2+]i that is consistent with the
initiation of a signaling cascade. However, on a molar basis, higher
concentrations of peptide were necessary to elicit
[Ca2+]i responses, as compared
with ApoE. This difference likely results from differences in receptor
affinity between the peptide and ApoE, a property generally seen when
comparing the effects of intact proteins with those of peptide ligands.
 View larger version (17K): [in a new window] |
Figure 5. [Ca2+]i measurements in macrophages exposed
to ApoE-mimetic peptide. Macrophages were stimulated with either 100 nM
ApoE-mimetic peptide (•) or 100 nM scrambled peptide ().
Approximately 60–70% of the macrophages demonstrated significant
increases in [Ca2+]i on stimulation with
ApoE. The data shown are representative of three individual experiments
each, using 25–30 cells each. The arrow indicates the time of peptide
addition. The details for [Ca2+]i measurement
are described in Materials and Methods.
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0.75 µM. In the presence of an 80-fold molar excess of ApoE, 79%
of the total 125I-labeled peptide binding was inhibited.
The binding of the peptide was also placed into competition with
increasing amounts of RAP (Fig. 6)
, which blocked 71% of the total
125I-labeled peptide binding with an IC50 of
<31 nM. These data suggest that the peptide sequence contains the
major determinant in the receptor binding of ApoE. It is noted that
intact ApoE competes for binding of the peptide somewhat more poorly
than the peptide itself. These results suggest that other secondary
determinants outside this region may contribute to receptor binding.
Alternatively, small conformational differences may exist for the free
peptide in comparison with that of this amino acid sequence as it
exists in the native protein. Such differences could also affect the
Kid for the binding reaction.
 View larger version (12K): [in a new window] |
Figure 6. Binding of 125I-labeled ApoE-mimetic peptide to macrophages
in the presence of ApoE and RAP. Macrophages were incubated with
125I-labeled ApoE-mimetic peptide (50 nM) in the presence
of ApoE ( ) or RAP () (31.25–4 µM). The data represent the
means of three studies each. Further details are in Materials and
Methods.
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ApoE, which is well known for its role in cholesterol metabolism, also demonstrates immunomodulatory properties, including suppression of lymphocyte proliferation, immunoglobulin synthesis, and neutrophil activation [1 2 3 4 5 6 7 ]. The mechanisms by which ApoE modulates immune responses remain undefined, although it is consistent with a receptor-mediated event [1 2 3 4 5 6 7 ]. The immunomodulatory effects of ApoE appear biologically relevant because ApoE-deficient animals have a variety of impaired immune responses including increased susceptibility to endotoxemia after challenge with lipopolysaccharide or K. pneumonia [5 ]. Macrophage functions have been implicated, given that ApoE-deficient animals have impaired delayed-type hypersensitivity responses and are particularly vulnerable to exposure to L. monocytogenes, an organism that requires normal macrophage function for clearance [6 ]. ApoE may serve a biologically relevant immunomodulatory role in atherogenesis, a process in which inflammation at the vascular wall may influence disease progression [see 17 for review].
The immunomodulatory properties of ApoE may also be relevant in the
central nervous system (CNS). In the CNS, ApoE is the primary
apolipoprotein synthesized intra-axially, and its secretion is markedly
up-regulated after injury. The endogenous CNS immune response is
characterized by activation of microglia, components of the
reticuloendothelial system present in the brain, resulting in the
secretion of inflammatory cytokines and reactive oxygen species. This
process has been implicated in a variety of acute and chronic
neurological diseases. Biologically relevant concentrations of ApoE
suppress microglial secretion of nitric oxide and inflammatory
cytokines such as TNF- [30
31
32
]. This is particularly
relevant to our studies given that microglia share many functional
characteristics of macrophages, including the expression of LRP
[18
].
ApoE-receptor interactions have generally been regarded as a mechanism for clearing ApoE-containing lipoproteins. In recent years, however, evidence has accumulated to suggest that endocytosis might not be the only function of this receptor system, and ApoE-receptor interactions might also mediate adaptive cellular responses through the initiation of signaling cascades. Several studies have suggested that ApoE initiates signal transduction in neurons [33 34 35 ]. A peptide derived from a tandem repeat of the ApoE receptor-binding region causes an increase in neuronal [Ca2+]i [36 ]. The biological relevance of these observations might be challenged, in view of the artificial nature of the tandem repeat construct and the reported supraphysiological concentrations of ApoE necessary to initiate an increase in intracellular Ca2+. Specifically, maximal intracellular Ca2+ was observed at an ApoE concentration of 30 µM, and the data were interpreted as an injury response [36 ]. The present study, however, suggests important regulatory properties for ApoE without being subject to these objections. Specifically, the concentration required for signal transduction was far lower with macrophages than reported for neurons [36 ]. Our results were not compromised by endotoxin contamination, because all preparations were assayed for endotoxin, and no significant contamination was observed. It should be noted, however, that we have never observed an increase in [Ca2+]i in macrophages with the addition of lipopolysaccharides (data not shown).
Although ApoE binds to a family of receptors, many of these receptors have not been definitively demonstrated on macrophages [15 16 17 23 24 25 ]. Indeed, we have directly examined macrophages to determine whether the LDL receptor or LRP accounts for the uptake of LDL [22 ]. These studies suggested that LRP accounts for all of the uptake of LDL by macrophages. LRP ligation by lactoferrin, lipoprotein lipase, thrombospondin, or Pseudomonas exotoxin A elevates intracellular Ca2+ by a pertussis toxin-sensitive G-protein-mediated pathway [19 20 37 ]. RAP and Ni2+ block both binding of ligands to LRP and signal transduction through LRP [19 20 ]. Thus, our current observations are most consistent with the hypothesis that ApoE initiates a signaling cascade in macrophages via specific interaction with LRP. We cannot, however, absolutely preclude the possibility of specific interactions with other members of the LDL receptor superfamily, such as the VLDL or ER-2. To rule out the possibility that our data may have been affected by endogenous murine ApoE secreted into the media, all cell culture media were changed, and cells were washed before the addition of ligands. Moreover, similar results were obtained with murine macrophages obtained from ApoE-deficient mice.
In summary, ApoE induces a signaling cascade in murine macrophages. This effect is initiated by the specific binding of ApoE to LRP and results in an increase in [Ca2+]i and synthesis of IP3 that is mediated by a pertussis toxin-sensitive G-protein. A 20-residue peptide derived from the ApoE receptor-binding site can initiate cellular signaling in a manner identical to that of ApoE, indicating that the ability of ApoE to induce signaling is independent of lipid binding. Moreover, the binding of the mimetic peptide to macrophages competes directly with intact protein, confirming that both ligands bind to the same receptor site. The inhibition of ApoE binding and signal transduction by RAP and Ni2+ implicates LRP as a target receptor for ApoE. The ability of ApoE to transduce a signal may be particularly relevant with respect to understanding its role in atherosclerosis, immune responses, and modulation of brain macrophage (microglia) functions after injury.
Received June 17, 2001; revised April 23, 2001; accepted April 24, 2001.
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2-macroglobulin receptor in Alzheimer and control postmortem human brain Mol. Chem. Neuropath. 18,153-160[Medline]2-macroglobulin (2M) receptors and the newly described 2M signaling receptor J. Biol. Chem. 269,18803-188062-macroglobulin-methylamine to the low density lipoprotein receptor related protein/ 2-macroglobulin receptor but not the 2-macroglobulin signaling receptor: evidence for a nickel effect on a region of 2-macroglobulin upstream of the receptor binding domain Biochemistry 36,12395-12399[Medline]2-macroglobulin (2M) with the low density lipoprotein receptor-related protein/2M receptor but not the 2M signaling receptor J. Clin. Invest. 97,1193-1203[Medline]2-macroglobulin receptor on murine peritoneal macrophages mediates the binding and catabolism of low density lipoprotein Arch. Biochem. Biophys. 326,39-47[Medline]-macroglobulin J. Biol. Chem. 267,18551-185572-macroglobulin-recognition site Biochem. J. 239,863-870This article has been cited by other articles:
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