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(Journal of Leukocyte Biology. 2001;70:677-683.)
© 2001 by Society for Leukocyte Biology

Apolipoprotein E and mimetic peptide initiate a calcium-dependent signaling response in macrophages

Uma K. Misra*, Charu L. Adlakha*, Govind Gawdi*, Michael K. McMillian{dagger}, Salvatore V. Pizzo* and Daniel T. Laskowitz{ddagger}

* Departments of Pathology,
{dagger} Pharmacology, and
{ddagger} 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.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apolipoprotein E (ApoE) is a 34-kDa cholesterol transport protein that also possesses immunomodulatory properties. In this study, we demonstrate that ApoE initiates a signaling cascade in murine peritoneal macrophages that leads to increased production of inositol triphosphate with mobilization of intracellular Ca2+ stores. This cascade is inhibited by pretreatment with receptor-associated protein and Ni2+, and it is mediated by a pertussis toxin-sensitive G protein. These properties are characteristic of signal transduction induced via ligand binding to the cellular receptor, lipoprotein receptor-related protein. A peptide derived from the receptor-binding region of ApoE also initiates signal transduction in a manner similar to that of the intact protein, suggesting that this isolated region is sufficient for signal transduction. The ApoE-mimetic peptide competed for binding with the intact protein, confirming that they both interact with the same site. ApoE-dependent signal transduction might play a role in mediating the functional properties of this lipoprotein.

Key Words: apolipoprotein E • receptor-associated protein • macrophage • lipoprotein receptor-related protein • signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apolipoprotein E (ApoE) is a multifunctional 299-amino-acid protein with a recognized role in cholesterol transport [1 ]. ApoE also possesses significant immunomodulatory properties in vitro, including the capacity to suppress lymphocyte proliferation, immunoglobulin synthesis, and neutrophil activation [1 2 3 4 ]. These immunomodulatory properties of ApoE appear to be biologically relevant, because ApoE-deficient animals have impaired immunity after exposure to Listeria monocytogenes and are more susceptible to endotoxemia after inoculation with lipopolysaccharide or Klebsiella pneumonia [5 6 ]. ApoE-deficient mice demonstrate differences in delayed-type hypersensitivity and immunoglobulin M (IgM) responses to antigen as compared with wild-type mice [7 ]. ApoE also modulates outcomes in human neurological diseases, although the mechanistic basis for this effect has not been defined [8 9 10 11 ]. These observations suggest that ApoE has important biological functions independent of its role in cholesterol metabolism.

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 {alpha} 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 {alpha}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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Brewer’s thioglycollate (TG) broth was purchased from Difco Laboratories (Baltimore, MD). RPMI 1640 medium, fetal bovine serum, Hanks’ balanced salt solution, and other cell culture reagents were purchased from Life Technologies, Inc. (Grand Island, NY). Bovine serum albumin (BSA), pertussis toxin, and HEPES were from Sigma Chemical Co. (St. Louis, MO). Fura-2/acetoxymethyl ester (AM) and 1,2-bis(O-aminophenyl-ethane-ethane)-Ni1N1N'1N'-tetraacetic acid (BAPTA)/AM were obtained from Molecular Probes (Eugene, OR). Myo-[2-3H]inositol (specific activity, 10–20 Ci/mmol) was from American Radiolabeled Chemicals, Inc. (St. Louis, MO). A plasmid containing the RAP cDNA was a gift from J. Herz (University of Texas, Southwestern, Dallas) and was used to produce RAP as previously described [21 ]. Thapsigargin was purchased from Biomol (Plymouth Meeting, PA). Human recombinant ApoE2 was obtained commercially from Panvera Corp. (Madison, WI) and was homogeneous as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. ApoE endotoxin levels were measured with the Kinetic-QCL Limulus amoebocyte lysate assay from BioWhittaker, Inc. (Walkersville, MD) and were determined to be <0.005 EU/mg. The 20-amino-acid ApoE-mimetic peptide (Ac-TEELRVRLASHLRKLRKRLL-amide), both with and without a tyrosine on the amino terminus, was synthesized by QCB Biochemicals (Hopkinton, MA) to a purity of 95%, as was a scrambled control peptide of identical size, amino acid composition, and purity. The ApoE-mimetic peptide containing a tyrosine was iodinated by American Radiolabeled Chemicals, Inc. (St. Louis, MO) (specific activity, 833 Ci/mmol). All amino termini were acetylated, and all carboxyl termini were blocked with an amide moiety. Peptides were reconstituted in sterile isotonic phosphate-buffered saline. All other reagents used were of the highest quality commercially available.

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 {gamma}-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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of ApoE on intracellular calcium levels
Cellular modulation of free cytoplasmic Ca2+ concentration is a ubiquitous signaling response. To determine whether ApoE can initiate signaling in macrophages, cells were treated with ApoE, and [Ca2+]i was measured as a function of time. In macrophages treated with human recombinant ApoE, [Ca2+]i levels increased two- to fourfold as compared with the baseline level (Fig. 1A ). The average [Ca2+]i levels of >200 cells were 95.33 ± 7.37 nM before stimulation and 180.25 ± 14.57 nM after ApoE treatment. The increase in [Ca2+]i on stimulation with ApoE was observed in 70–80% of the cells examined. ApoE-induced increases in [Ca2+]i were heterogeneous, asynchronous, and either oscillatory or sustained. The [Ca2+]i increase caused by ApoE stimulation was found to be dependent on the ApoE concentration (Fig. 1B) .



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  		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.

 
Intracellular Ca2+ increases can be caused by either Ca2+ release from intracellular stores or extracellular Ca2+ influx through gated Ca2+ channels [27 ]. To assess the source of [Ca2+]i after ApoE treatment, we attempted to stimulate cells after depleting endoplasmic reticulum Ca2+ stores with thapsigargin (TG) (Fig. 1C) . The addition of ApoE to TG-treated cells did not affect [Ca2+]i levels, suggesting that the increase in [Ca2+]i observed in ApoE-stimulated macrophages is caused by release from intracellular stores and not by influx via a Ca2+ channel.

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.



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  		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 ({circ}) 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.

 
Effect of pertussis toxin on ApoE-induced signal transduction
To characterize the G-protein through which ApoE-induced signaling occurs, macrophages were pretreated with pertussis toxin before the addition of ApoE. The increase in IP3 levels caused by the ApoE exposure was completely inhibited by pertussis toxin (Fig. 2A) . This result demonstrated that the ApoE-induced generation of IP3 is coupled to a pertussis toxin-sensitive G-protein. The ApoE effect on [Ca2+]i levels was also completely blocked by pertussis toxin pretreatment, indicating that ApoE-induced signaling occurs solely via a pertussis toxin-sensitive pathway (Fig. 3 ).



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  		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) ({circ}) 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.

 
Effect of Ni2+ and RAP on ApoE-induced signal transduction
Previous studies have demonstrated that ApoE binds to LRP and is then internalized [16 23 24 25 ]. Additionally, binding of LRP to ligands such as lactoferrin, Pseudomonas exotoxin A, lipoprotein lipase, and thrombospondin initiates a signaling cascade associated with the generation of second messengers [19 20 21 22 ]. To investigate the possibility that LRP was involved in the signal cascade induced by ApoE, macrophages were preincubated with RAP and Ni2+ before stimulation with ApoE. RAP is a 39-kDa protein that blocks the binding of all known ligands to LRP [28 ]. Ni2+ also blocks ligand interactions with LRP [20 29 ]. Preincubation with either RAP or Ni2+ markedly attenuated the [Ca2+]i increases associated with subsequent exposure to ApoE (Fig. 4 ). These results are consistent with the hypothesis that ApoE induces a signaling cascade via specific interaction with LRP [19 20 ].



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  		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) ({square}), or NiCl2 (10 mM) ({triangleup}) 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.

 
Effect of ApoE-mimetic peptide on intracellular calcium levels
To better understand the region of ApoE responsible for the initiation of signaling, we generated an ApoE-mimetic peptide consisting of the ApoE receptor-binding region spanning residues 130–149. Stimulation of macrophages with the ApoE-mimetic peptide resulted in a two- to threefold increases in [Ca2+]i, whereas the scrambled control peptide had no effect (Fig. 5 ). This increase in [Ca2+]i was observed in ~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.



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  		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 ({circ}). 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.

 
Binding competition of ApoE-mimetic peptide with ApoE
Having determined that the ApoE-mimetic peptide can induce signaling, it was necessary to confirm that the peptide and the protein were binding to the same site. Before performing competition studies, the dissociation constant of the peptide was determined by direct binding studies of the radiolabeled peptide on macrophages. The binding data were analyzed by a nonlinear direct fit to the one-site-binding equation, resulting in a dissociation constant of approximately 50 nM (data not shown). The binding of radiolabeled peptide then competed with increasing amounts of ApoE (Fig. 6 ). ApoE competed with the peptide for binding to peritoneal macrophages with a 50% inhibitory concentration (IC50) of ~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.



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  		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 ({blacksquare}) or RAP ({square}) (31.25–4 µM). The data represent the means of three studies each. Further details are in Materials and Methods.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrate that binding to macrophage cell surface receptors by human recombinant ApoE initiates signaling events associated with dose-dependent increases in IP3 and [Ca2+]i. The ApoE-induced increase in [Ca2+]i is pertussis toxin sensitive. A 20-residue peptide derived from the receptor-binding region of ApoE is sufficient to cause similar changes in macrophage [Ca2+]i, indicating that the initiation of signaling is receptor mediated and independent of lipid binding. Additionally, the ability of RAP and Ni2+ to block both receptor binding and signal transduction of ApoE suggests that ApoE induces signaling via interactions with LRP.

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-{alpha} [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.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health grants NS368087-01A2, K08NS01949, R37HL24066, and RO3 AG16507-01. Dr. Laskowitz is a Paul Beeson Physician Faculty Scholar.

Received June 17, 2001; revised April 23, 2001; accepted April 24, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Avila, E. M., Holdsworth, G., Sasaki, N., Jackson, R. L., Harmony, J. A. (1982) Apoprotein E suppresses phytohemagglutinin-activated phospholipid turnover in peripheral blood mononuclear cells J. Biol. Chem. 257,5900-5909[Abstract/Free Full Text]
  2. Curtiss, L. K., Edgington, T. S. (1981) Immunoregulatory plasma low density lipoprotein: the biologic activity and receptor-binding specificity is independent of neutral lipids J. Immunol. 126,1382-1386[Abstract]
  3. Hui, D. Y., Harmony, T. L., Innerarity, R. W. (1980) Immunoregulatory plasma lipoproteins: role of apoprotein E and apoprotein B J. Biol. Chem. 255,11775-11781[Abstract/Free Full Text]
  4. Pepe, M. G., Curtiss, L. K. (1986) Apolipoprotein E is a biologically active constituent of the normal immunoregulatory lipoprotein, LDL-In J. Immunol. 136,3716-3723[Abstract]
  5. Roselaar, S. E., Daugherty, A. (1998) Apolipoprotein E-deficient mice have impaired innate immune responses to Listeria monocytogenes in vivo J. Lipid Res. 39,1740-1743[Abstract/Free Full Text]
  6. deBont, N., Netea, M. G., Demacker, P. N., Verschueren, I., Kullberg, B. J., vanDijk, K. W., vanderMeer, J. W., Stalenhoef, A. F. (1999) Apolipoprotein E knock-out mice are highly susceptible to endotoxemia and Klebsiella pneumoniae infection J. Lipid Res. 40,680-685[Abstract/Free Full Text]
  7. Laskowitz, D. T., Lee, D. M., Schmechel, D., Staats, H. F. (2000) Altered immune responses in apolipoprotein E-deficient mice J. Lipid Res. 41,613-620[Abstract/Free Full Text]
  8. Corder, E. H., Saunders, A. M., Strittmatter, W. J., Schmechel, D. E., Gaskell, P. C., Small, G. W., Roses, A. D., Haines, J. L., Pericak-Vance, M. A. (1993) Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families Science 261,828-829[Free Full Text]
  9. Alberts, M. J., Graffagnino, C., McClenny, C., DeLong, D., Strittmatter, W. J., Saunders, A. M., Roses, A. D. (1995) ApoE genotype and survival from intracerebral haemorrhage Lancet 346,575[Medline]
  10. Teasdale, G. M., Nicoll, J. A., Murray, G., Fiddes, M. (1997) Association of apolipoprotein E polymorphism with outcome after head injury Lancet 350,1069-1071[Medline]
  11. Laskowitz, D. T., Sheng, H., Bart, R. D., Joyner, K. A., Roses, A. D., Warner, D. S. (1997) Apolipoprotein E-deficient mice have increased susceptibility to focal cerebral ischemia J. Cereb. Blood Flow Metab. 17,753-758[Medline]
  12. Wetterau, J. R., Aggerbeck, L. P., Rall, S. C., Jr, Weisgraber, K. H. (1988) Human apolipoprotein E3 in aqueous solution. I. Evidence for two structural domains J. Biol. Chem. 263,6240-6248[Abstract/Free Full Text]
  13. Weisgraber, K. H., Innerarity, T. L., Harder, K. J., Mahley, R. W., Milne, R. W., Marcel, L., Sparrow, J. T. (1983) The receptor-binding domain of human apolipoprotein E: monoclonal antibody inhibition of binding J. Biol. Chem. 258,12348-12354[Abstract/Free Full Text]
  14. Innerarity, T. L., Friedlander, E. J., Rall, S. C., Jr, Weisgraber, K. H., Mahley, R. W. (1983) The receptor-binding domain of human apolipoprotein E: binding of apolipoprotein E fragments J. Biol. Chem. 258,12341-12347[Abstract/Free Full Text]
  15. Knott, T. J., Pease, R. J., Powell, L. M., Wallis, S. C., Rall, S. C., Jr, Innerarity, T. L., Blackhart, B., Taylor, W. H., Marcel, Y., Milne, R. (1986) Complete protein sequence and identification of structural domains of human apolipoprotein B Nature 323,734-738[Medline]
  16. Beisiegel, U., Weber, W., Ihrke, G., Herz, J., Stanley, K. K. (1989) The LDL-receptor-related protein, LRP, is an apolipoprotein E-binding protein Nature 314,162-164
  17. Curtiss, L. K., Boisvert, W. A. (2000) Apolipoprotein E and atherosclerosis Curr. Opin. Lipidol. 11,243-251[Medline]
  18. Tooyama, I., Kawamata, T., Akiyama, H., Moestrup, S. K., Gliemann, J., McGeer, P. L. (1993) Immunohistochemical study of {alpha}2-macroglobulin receptor in Alzheimer and control postmortem human brain Mol. Chem. Neuropath. 18,153-160[Medline]
  19. Misra, U. K., Chu, T. C. C., Gawdi, G., Pizzo, S. V. (1994) The relationship between low density lipoprotein-related protein/{alpha}2-macroglobulin ({alpha}2M) receptors and the newly described {alpha}2M signaling receptor J. Biol. Chem. 269,18803-18806
  20. Odom, A. R., Misra, U. K., Pizzo, S. V. (1997) Nickel inhibits biding of {alpha}2-macroglobulin-methylamine to the low density lipoprotein receptor related protein/ {alpha}2-macroglobulin receptor but not the {alpha}2-macroglobulin signaling receptor: evidence for a nickel effect on a region of {alpha}2-macroglobulin upstream of the receptor binding domain Biochemistry 36,12395-12399[Medline]
  21. Howard, G. C., Misra, U. K., DeCamp, D. L., Pizzo, S. V. (1996) Altered interaction of cisdichlorodiammineplatinum(II)-modified {alpha}2-macroglobulin ({alpha}2M) with the low density lipoprotein receptor-related protein/{alpha}2M receptor but not the {alpha}2M signaling receptor J. Clin. Invest. 97,1193-1203[Medline]
  22. Wu, S. W., Pizzo, S. V. (1996) Low density lipoprotein receptor-related protein/{alpha}2-macroglobulin receptor on murine peritoneal macrophages mediates the binding and catabolism of low density lipoprotein Arch. Biochem. Biophys. 326,39-47[Medline]
  23. Huettinger, M., Retzek, H., Hermann, M., Goldenberg, H. (1992) Lactoferrin specifically inhibits endocytosis of chylomicron remnants but not {alpha}-macroglobulin J. Biol. Chem. 267,18551-18557[Abstract/Free Full Text]
  24. van Dijk, M. C. M., Ziere, G. J., Boers, W., Linthorst, C., Bijsterbosch, M. K., Van Berkel, J. C. (1991) Recognition of chylomicron remnants and ß-migrating very-low-density lipoproteins by the remnant receptor of parenchymal liver cells is distinct from the liver {alpha}2-macroglobulin-recognition site Biochem. J. 239,863-870
  25. Ji, Z. S., Mahley, R. W. (1994) Lactoferrin binding to heparan sulfate proteoglycans and the LDL receptor-related protein: further evidence supporting the importance of direct binding of remnant lipoproteins to HSPG Arterioscler. Thromb. 14,2025-2032[Abstract/Free Full Text]
  26. Berridge, M. D. (1983) Rapid accumulation of inositol triphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol Biochem. J. 212,709-724
  27. Berridge, M. D. (1995) Capacitative calcium entry Biochem. J. 312,1-11
  28. Strickland, D. K., Ashcom, J. D., Williams, S., Burgess, W. H., Migliorini, M., Argraves, W. S. (1990) Sequence identity between the alpha 2-macroglobulin receptor and low density lipoprotein receptor-related protein suggests that this molecule is a multifunctional receptor J Biol. Chem. 265,17401-17404[Abstract/Free Full Text]
  29. Hussain, M. M., Kanch, R. K., Tulenko, T. N. (1995) Nickel is a specific inhibitor for the binding of activated alpha 2-macroglobulin to the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor Biochemistry 34,16074-16081[Medline]
  30. Laskowitz, D. T., Goel, S., Bennett, E. R., Matthew, W. D. (1997) Apolipoprotein E suppresses glial cell secretion of TNF alpha J. Neuroimmunol. 76,70-74[Medline]
  31. Laskowitz, D. T., Matthew, W. D., Bennett, E. R., Schmechel, D. E., Herbstreith, M. H., Goel, S., McMillain, M. K. (1998) Endogenous apolipoprotein E suppresses LPS-stimulated microglial nitric oxide production Neuroreport 9,615-618[Medline]
  32. Barger, S. W., Harmon, A. D. (1997) Microglial activation by Alzheimer amyloid precursor protein and modulation by apolipoprotein E Nature 388,878-881[Medline]
  33. Bellosta, S., Nathan, B. P., Orth, M., Dong, L. M., Mahley, R. W., Pitas, R. E. (1995) Stable expression and secretion of apolipoproteins E3 and E4 in mouse neuroblastoma cells produces differential effects on neurite outgrowth J. Biol. Chem. 270,27063-27071[Abstract/Free Full Text]
  34. Holtzman, D. M., Pitas, R. E., Kilbridge, J., Nathan, B., Mahley, R. W., Bu, G., Schartz, A. L. (1995) Low density lipoprotein receptor-related protein mediates apolipoprotein E-dependent neurite outgrowth in a central nervous system-derived neuronal cell line Proc. Natl. Acad. Sci. USA 92,9480-9484[Abstract/Free Full Text]
  35. Nathan, B. P., Bellosta, S., Sanan, D. A., Weisgraber, K. H., Mahley, R. W., Pitas, R. E. (1994) Differential effects of apolipoproteins E3 and E4 on neuronal growth in vitro Science 264,850-852[Abstract/Free Full Text]
  36. Wang, X. S., Gruenstein, E. J. (1997) Rapid elevation of neuronal cytoplasmic calcium by apolipoprotein E peptide Cell Physiol 173,73-83
  37. Goretzki, L., Mueller, B. M. (1998) Low-density-lipoprotein-receptor-related protein (LRP) interacts with a GTP-binding protein Biochem. J. 336,381-386



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