Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Dec 24.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2011 Feb 24;31(5):1160–1168. doi: 10.1161/ATVBAHA.111.222745

APOLIPOPROTEIN E (APOE) INDUCES ANTI-INFLAMMATORY PHENOTYPE IN MACROPHAGES

Daniel Baitsch 1,*, Hans H Bock 2,3,*, Thomas Engel 4, Ralph Telgmann 4, Carsten Müller-Tidow 5, Georg Varga 6, Martine Bot 7, Joachim Herz 2,8, Horst Robenek 4, Arnold von Eckardstein 9, Jerzy-Roch Nofer 1,10
PMCID: PMC3529398  NIHMSID: NIHMS425470  PMID: 21350196

Abstract

Objectives

Apolipoprotein E (apoE) exerts potent anti-inflammatory effects. We here investigated the effect of apoE on the functional phenotype of macrophages.

Methods and Results

Human apoE receptors VLDL-R or apoER2 were stably expressed in RAW264.7 mouse macrophages. In these cells apoE downregulated markers of the pro-inflammatory M1 phenotype (iNOS, IL-12, MIP-1α), but upregulated markers of the anti-inflammatory M2 phenotype (arginase-I, SOCS3, IL-1RA). In addition, M1 macrophage responses (migration, generation of reactive oxygen species, antibody-dependent cell cytotoxicity, phagocytosis) as well as poly(I:C)- and/or IFN-γ-induced production of pro-inflammatory cytokines, COX-2 expression, and activation of NF-κB, IκB and STAT1 were suppressed in VLDL-R- or apoER2-expressing cells. Conversely, the suppression of M2 phenotype and the enhanced response to poly(I:C) were observed in apoE-producing bone marrow macrophages derived from VLDL-R-deficient mice, but not wild type or LDL receptor-deficient mice. The modulatory effects of apoE on macrophage polarization were inhibited in apoE receptor-expressing RAW264.7 cells exposed to SB220025, a p38MAP kinase inhibitor, and PP1, a tyrosine kinase inhibitor. Accordingly, apoE induced tyrosine kinase-dependent activation of p38MAP kinase in VLDL-R- or apoER2-expressing macrophages. Under in vivo conditions, apoE−/− mice transplanted with apoE-producing wild-type bone marrow showed increased plasma IL-1RA levels and peritoneal macrophages of transplanted animals were shifted to the M2 phenotype (increased IL-1RA production and CD206 expression).

Conclusion

ApoE signaling via VLDL-R or apoER2 promotes macrophage conversion from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype. This effect may represent a novel anti-inflammatory activity of apoE.

Keywords: Apolipoprotein E (apoE), macrophage, inflammation, atherosclerosis

INTRODUCTION

Apolipoprotein E (apoE) is a major protein component of very low density lipoproteins (VLDL) and high density lipoproteins (HDL). Observations in human carriers of apoE variants and in apoE-deficient mice suggest an important role of this apolipoprotein in preventing atherosclerosis [1]. The anti-atherogenic effects of apoE are usually attributed to its ability to regulate VLDL production and to facilitate hepatic clearance of VLDL and chylomicron remnants [1]. However, apoE protects against atherosclerosis even in the absence of measurable effects on plasma lipoprotein metabolism. For instance, transgenic expression of apoE in arterial wall reduces the formation of atherosclerotic lesions without affecting plasma lipoprotein profile [2,3]. Conversely, enhanced atherosclerosis is observed in apoE+/+ animals transplanted with bone marrow from apoE-deficient mice [4]. These results suggest that apoE exerts local anti-atherogenic effects in the arterial wall that are independent from regulation of plasma lipoprotein metabolism.

An increasing body of evidence suggests that atherosclerosis is a chronic inflammatory disease. Macrophages assume a critical role in the initiation and the perpetuation of intravascular inflammation through their ability to produce an array of cytokines and chemokines, to generate reactive oxygen species, and to process and present antigens to CD4+ T cells. Importantly, macrophages represent a heterogeneous cell population that is distinctly activated by various microenvironmental signals [5–8]. Pro-atherogenic factors such as Th1 cytokines (e.g. interferon-γ (IFNγ), interleukin (IL) 1β (IL-1β)) and Toll-like receptor (TLR) ligands (e.g. poly(I:C), lipopolysaccharide (LPS)) induce a “classical” activation profile (M1) characterized by enhanced production of pro-inflammatory cytokines, expression of MHC class II molecules, and generation of free radicals including inducible NO synthase (iNOS)-derived nitric oxide (NO). In contrast, exposure of macrophages to Th2 cytokines (IL-4, IL-13) promotes the “alternative” activation profile (M2) comprising secretion of anti-inflammatory factors (e.g. interleukin-1 receptor antagonist (IL-1RA)), expression of the mannose receptor CD206 and/or hemoglobin receptor CD163, and reduced NO production due to the upregulation of arginase-I (Arg-I) activity, which converts arginine to ornithine and urea. In addition, alternatively activated macrophages demonstrate higher phagocytic capacity and reduced motility and cytotoxic effects.

Whereas the presence of both M1- and M2-polarized macrophages in the arterial wall and the progressive M2 to M1 switch during atherosclerotic lesion development have been recently documented [9], the mechanisms contributing to the generation of distinct macrophage phenotypes in context of atherosclerosis remain unclear. In the present study, we report that apoE exerts anti-inflammatory activity by switching the macrophage phenotype from M1 to M2 in a process involving signaling via the very low density lipoprotein receptor (VLDL-R) or the apoE receptor-2 (apoER2).

METHODS

Bone marrow (BM) transplantation

Homozygous apoE−/− and LDL receptor-deficient (LDL-R−/−) mice, both on a C57BL/6J background, and wild-type (WT) C57BL/6J mice were obtained from the Charles River Laboratories (Sulzfeld, Germany). VLDL-R−/− and apoER2−/− mice were generated as described previously [10,11]. To induce bone marrow (BM) aplasia, apoE−/− mice (12 animals) were exposed to a single dose of 11 Gy total body irradiation. BM (106 cells) isolated by flushing femurs from apoE−/− and WT mice were administered i.v. Chimerism was assessed in leukocyte DNA by PCR.

ApoE receptor cloning and transfections

Human endothelial and brain DNA were reverse transcribed and subjected to PCR with VLDL-R and apoER2 gene-specific primers, respectively. PCR products corresponding to VLDL-R and apoER2 were ligated into expression vector pBK-CMV. RAW264.7 cells were stably transfected by electroporation. Positive clones were isolated and identified by Western blot.

Analytical procedures

Expression of phenotype-specific markers and phosphorylation of protein kinases p38MAPK and Akt were examined by Western blot. Production of cytokines, prostaglandin E2 (PGE2) and NO were examined with ELISA or EIA. ApoE binding, CD206 and MHC-II cell surface expression and fluorescent latex beads uptake for estimation of phagocytosis were investigated by flow cytometry. Transcription factor activity was examined by luciferase reporter assays using gene vectors p(κB)5-Luc and p(GAS)4-Luc. NO and H2O2 production were assessed using fluorescence spectroscopy. Production of urea and antibody-dependent cell cytotoxicity (ADCC) were investigated by light spectrometry.

Statistical analysis

Data are presented as means ± S.D. from at least three separate experiments or as results representative for at least three repetitions, unless indicated otherwise. Comparisons were performed with two-tailed Student t-test. Detailed Methods can be found online (http://atvb.ahajournals.org).

RESULTS

Generation of stable transfectants expressing functional VLDL-R- or apoER2

Two stable cell lines were generated by transfection of RAW264.7 macrophages with plasmids encoding human variants of VLDL-R or apoER2 expressed in the vasculature. Each cell line produced proteins with expected molecular weights cross-reacting with antibodies either against VLDL-R or apoER2, displayed specific binding of apoE, and produced no endogenous apoE (for details see Supplementary Materials; http://atvb.ahajournals.org).

ApoE induces typical characteristics of alternative activation in VLDL-R- or apoER2-expressing RAW264.7 macrophages

To investigate whether apoE drives macrophages towards the M2 phenotype, RAW264.7 cells expressing VLDL-R or apoER2 were incubated for 24 h with apoE, and the expression of M1 and M2 polarization markers was investigated. Fig. 1A demonstrates that, relative to control cells, the expression of iNOS, a hallmark of classically activated macrophages [5,6], was down-regulated in VLDL-R- or apoER2-expressing macrophages in the presence of apoE. By contrast, cells expressing VLDL-R or apoER2 displayed up-regulated arginase-I, FIZZ1/RELM, and SOCS3, three markers of alternative macrophage activation, in response to apoE [6–8]. Consistent with these observations, apoE decreased the enzymatic activity of iNOS, as inferred from reduced nitrite/nitrate generation, whereas the production of urea, an arginase-I product, was increased (Fig. 1B). The latter effect was concentration-dependent and reached the maximum at 5 μg/mL apoE. In addition, preincubation of VLDL-R- or apoER2-expressing macrophages with apoE reduced the steady-state production of M1 cytokines IL-12 and MIP-1α, whereas the production of M2 cytokines IL-1RA and G-CSF was enhanced in a concentration-dependent fashion indicating that apoE shifts the balance from a pro-inflammatory towards an anti-inflammatory cytokine profile (Fig. 1C). Endotoxin contamination did not account for the modulatory effects of apoE on macrophage functional phenotype (see Supplementary Materials; http://atvb.ahajournals.org.)

Figure 1. ApoE induces macrophage M2 polarization markers in VLDL-R- or apoER2-expressing RAW264.7 cells.

Figure 1

Open in a new tab

VLDL-R, apoER2 or wild-type RAW264.7 cells were cultured for 24h with or without apoE (5μg/mL) or with increasing concentrations of apoE. A. Total cell lysates were separated by SDS-PAGE and immunoblotted with antibodies against iNOS, arginase-1, FIZZ1/RELM, and SOCS3. Data are representative for 3 independent experiments. B. Cell culture media were collected and concentrations of nitrite and nitrate (products of iNOS) and urea (product of arginase-1) were determined by fluorimetric or photometric assays, respectively. Shown are results from 3 independent experiments. C and D. Concentrations of M1 (MIP-1α, IL-12) and M2 (IL-1RA, G-CSF, IL-4, IL-13) cytokines were determined by ELISA. IL-13Rα2 was co-incubated with apoE for 24h at concentration of 0.1 μg/mL. Shown are results from 3 to 5 independent experiments. * - p<0.05; § - p<0.01; # - p<0.001, −apoE vs. +apoE).

As IL-4 and IL-13, two cytokines secreted by macrophages, are typical inducers of the alternative macrophage phenotype [6–8], we next examined, whether the activation pattern seen in RAW264.7 cells expressing VLDL-R or apoER2 is a direct effect of apoE or whether it is conferred via intermediate products. As shown in Fig. 1D, exposure of macrophages to apoE failed to induce IL-4 production, but led to a moderate release of IL-13 into cell media. However, recombinant mouse IL13 receptor α2 (IL-13Rα2), which was previously shown to abrogate cytokine effects both under in vitro and in vivo conditions [12,13], failed to influence apoE-stimulated IL-1RA and G-CSF production in VLDL-R- or apoER2-expressing cells, which argues against the notion that apoE drives the M2 polarization via the IL-13-mediated paracrine loop.

ApoE alters functional phenotype in VLDL-R- or apoER2-expressing RAW264.7 macrophages

As alternative macrophage activation is accompanied by changes of cell function such as decreased cell motility and cytotoxicity and increased phagocytosis [5–7], we next investigated the propensity of apoE to modulate the macrophage functional phenotype in RAW264.7 cells expressing VLDL-R or apoER2. As shown in Fig. 2A, the induction of chemotaxis by two common leukocyte chemoattractants, M-CSF and fMLP, was equally effective in control and apoE receptor-expressing RAW264.7 macrophages. However, these migratory responses were diminished in the presence of apoE only in cells expressing VLDL-R or apoER2. Similarly, cytotoxic effects of macrophages, as determined by hemoglobin release from opsonized erythrocytes, were attenuated in the presence of apoE in VLDL-R- or apoER2-expressing cells but not in control cells (Fig. 2B). This was likely a consequence of reduced ROS generation, as the preincubation of RAW264.7 macrophages with apoE decreased the ROS generation rate in VLDL-R- and apoER2-expressing cells, while no inhibitory effects were noted in control cells (Fig. 2C). In addition, pretreatment with apoE consistently increased the number of VLDL-R- or apoER2-expressing macrophages phagocytosing FITC-latex beads (Fig. 2D). By contrast, apoE failed to affect phagocytosis in control RAW264.7 macrophages.

Figure 2. ApoE induces M2 functional phenotype in VLDL-R- or apoER2-expressing RAW264.7 cells.

Figure 2

Open in a new tab

VLDL-R, apoER2 or WT RAW264.7 macrophages were incubated with apoE (5 μg/mL) for 24h. A. Cell suspension (2.0 × 105/mL) was exposed to chemoattractans fMLP (0.1 μmol/L) or M-CSF (0.1 μmol/L) for 2h. Cells emigrated through the insert membrane were fixed, stained and quantified as described in Methods. Shown are results from 3 independent experiments. B. Adhering cells were incubated for 2h with opsonized erythrocytes and hemoglobin released to the media was quantified as described in Methods. Shown are results from 3 independent experiments. C. Cell suspensions (5.0 × 105/mL) were loaded with H2DCF-DA and DCF fluorescence reflecting H2O2 concentration was monitored fluorimetrically for 20 min. Original tracing curves from one representative experiment out of three were superimposed for comparison (left panels). Right panel: comparison of ROS production rate in cells treated or not with apoE. D. Adhering cells were co-incubated with apoE and FITC-coupled latex beads for 18h. The single cell fluorescence was analyzed by flow cytometry. Note fluorescence peaks corresponding to macrophage populations ingesting one, two or more beads. Original tracing curves from one representative experiment out of three were superimposed for comparison (left panels). Right panel: comparison of latex beads ingestion stimulated by apoE in macrophages. * - p<0.05; § - p<0.01; # - p<0.001, −apoE vs. +apoE.

ApoE inhibits the pro-inflammatory response of RAW264.7 macrophages to IFNγ and poly(I:C)

Alternatively activated macrophages are characterized by impaired response to M1 stimuli such as Th1 cytokines and TLR ligands [6–8]. Therefore, we next assessed the ability of RAW264.7 macrophages to respond to poly(I:C) and/or IFNγ stimulation with cyto/chemokine production and activation of transcription factors NF-κB- or STAT1. ApoE alone failed to induce cytokine or chemokine production. However, following exposure to apoE, VLDL-R- or apoER2-expressing macrophages were significantly suppressed with respect to production of pro-inflammatory cytokines/chemokines (IL-12, TNFα, MCP-1, IL-6) as compared to control RAW264.7 cells (Fig. 3A). Moreover, the expression of cyclooxygenase-2 (COX-2), a marker of pro-inflamatory macrophage activation, and the release of a COX-2 product, PGE2, in response to IFNγ and poly(I:C) were both suppressed in the presence of apoE in VLDL-R- or apoER2-expressing but not in control macrophages (Fig. 3B). The reduced pro-inflammatory response to M1 stimuli was further reflected at the transcriptional level by impaired upregulation of poly(I:C)-induced NF-κB activation or IFNγ–inducedSTAT1 activation in apoE -pretreated VLDL-R or apoER2-expressing macrophages transiently transfected with reporter plasmids containing either NF-κB- or STAT1-responsive promoters (Fig. 3C). At the pre-transcriptional level, degradation and phosphorylation of IκB, a component of the NF-κB complex, as well as phosphorylation of STAT1, which both precede the initiation of transcription by NF-κB- or STAT1, respectively, were abolished in VLDL-R or apoER2-expressing macrophages in the presence of apoE (Fig. 3D). By contrast, both NF-κB/STAT1 activation and IκB/STAT-1 phosphorylation in response to poly(I:C) or IFNγ remained unaffected by apoE in control RAW264.7 macrophages (not shown).

Figure 3. ApoE suppresses poly(I:C)- and IFN-γ-induced inflammatory response in VLDL-R- or apoER2-expressing RAW264.7 cells.

Figure 3

Open in a new tab

VLDL-R, apoER2 or WT RAW264.7 macrophages were incubated with apoE (5 μg/mL) for 24h. A and B. Cells were stimulated for further 24h with poly(I:C) (10 ng/mL) and IFN-γ (50 ng/mL), supernatants were collected and analyzed by ELISA for cyto/chemokine content (C) or by EIA for PGE2 concentration (D, lower panel). Shown are results from stimulated cells representative for 3 to 5 independent experiments. * - p<0.05; § - p<0.01; # - p<0.001, stimulated vs. unstimulated cells. ApoE alone did not affect cyto/chemokine or PGE2 concentrations. Cell lysates were immunoblotted and probed with anti-COX-2 antibody (B, upper panel). Shown is one representative result out of three. Lower panel: densitometric analysis of blots. C. Cells were transfected with p(κB)5-Luc or p(Gas)-Luc reporter plasmids, stimulated for 24h with, respectively, poly(I:C) (10 ng/mL) or IFN-γ (50 ng/mL) with or without apoE, and analyzed for luciferase. Luminescence level in un-stimulated cells was set as 1. ApoE alone did not change luminescence levels. Shown are results from 3 to 4 independent experiments. * - p<0.05; § - p<0.01; −apoE vs. +apoE. D. Cells were exposed to poly(I:C) or IFN-γ for indicated times and lysates were probed with antibodies against total (t) and phosphorylated (p) isoforms of IκB and STAT1. Shown are blots representative for 3 to 5 independent experiments.

VLDL-R-deficient BM macrophages display enhanced pro-inflammatory phenotype

We next examined whether the effects of apoE on macrophage phenotype are also observed in normal primary macrophages, which do not overexpress apoE receptors. Initial studies documented the expression of VLDL-R but not apoER2 in BM-derived murine macrophages (Fig. 4A). In contrast to RAW264.7 cells, BM macrophages produced endogenous apoE and the amounts of apolipoprotein released into media within a 24h incubation period were comparable in cells derived from wild-type, VLDL-R−/−, apoER2−/− and LDL-R−/− mice (Fig. 4B). As shown in Fig. 4B, the expression of M2 polarization markers (arginase-1, SOCS3) was reduced after 24h incubation in cells obtained from VLDL-R−/− but not apoER−/− mice. In addition, VLDL-R-deficient BM macrophages were characterized by reduced production of urea as well as secretion of M2 cytokines (IL-1RA, IL-13; Fig. 4C). By contrast, the M1 functional phenotype was augmented in VLDL-R−/− macrophages as reflected by the increased production of inflammatory cytokines/chemokines (MCP-1, IL-6), the COX-2 expression and the release of PGE2 in response to stimulation with poly(I:C) (Fig. 4D and E). No differences with regard to M1/M2 polarization were noted between BM macrophages derived from wild type and LDL-R−/− deficient mice (Fig. 4B–E).

Figure 4. Bone marrow (BM) macrophages derived from VLDL-R−/− mice show enhanced pro-inflammatory phenotype.

Figure 4

Open in a new tab

BM macrophages were incubated for 24h in serum-free medium containing 0.1% (v/v) albumin. A. mRNA levels were assessed by RT-PCR. Shown are representative agarose gels of amplified VLDL-R, apoER2 and GAPDH DNA fragments. Cerebellum (Cer) mRNA was taken as positive control. B. Concentrated cell culture media and cell lysates were subjected to Western blot using anti-apoE, anti-arginase-1 and anti-SOCS-3 antibodies. Urea was determined by photometric assay C. Cytokine concentrations were determined by ELISA. D and E. Cells were stimulated for further 24h with poly(I:C) (10 ng/mL), supernatants were collected and analyzed by ELISA for cyto/chemokine content (D) or by EIA for PGE2 concentration (E, right panel). Cell lysates were immunoblotted with anti-COX-2 antibody and blots were analyzed by densitometry (E, right panel). Data represent mean ± SD from 3 independent experiments, each in duplicate. * - p<0.05; § - p<0.01, WT vs. VLDL-R−/− cells.

ApoE signals via p38 mitogen-activated protein kinase (p38MAPK) and tyrosine kinase

Binding of apoE to VLDL-R or apoER2 receptors results in the recruitment of intracellular adapter protein disabled-1 (Dab1) and culminates in the activation of protein kinase Akt [14]. However, RAW264.7 cells do not express Dab1 (not shown) and, accordingly, apoE failed to produce Akt phosphorylation both in control and apoE receptor-expressing macrophages (Fig. 5A). By contrast, time-dependent increase in p38MAPK phosphorylation upon incubation with apoE was observed in macrophages expressing VLDL-R or apoER2, but not in control cells (Fig. 5A). The apoE-induced p38MAPK phosphorylation was substantially reduced after preincubation of macrophages with PP1 (10.0 μmol/L), a potent inhibitor of the Src family of tyrosine kinases (Fig. 5B). To assess the involvement of p38MAPK and tyrosine kinase activation in the modulatory effects of apoE on macrophage M1/M2 phenotype, the apoE-induced production of IL-1RA was examined in macrophages preincubated with PP1 or SB203580, a specific inhibitor of p38MAPK. As shown in Fig. 5C, both inhibitors abolished apoE-induced production of M2 cytokines in VLDL-R- or apoER2-expressing RAW264.7 cells.

Figure 5. ApoE induces tyrosine kinase-dependent p38MAPK activation in VLDL-R- or apoER2-expressing RAW264.7 cells.

Figure 5

Open in a new tab

A. VLDL-R, apoER2 or WT RAW264.7 macrophages were incubated with apoE (5 μg/mL) for indicated times. Cell lysates were immunobloted and probed with antibodies against total (t) and phosphorylated (p) isoforms of Akt and p38MAPK. Results are representative for 3 experiments. B. Cells expressing apoE receptors were pretreated for 0.5h min with PP1 (10 μmol/L) and exposed to apoE (5 μg/mL) for 1h. p38MAPK phosphorylation was analyzed as described above. Shown are results representative for 2 experiments C. VLDL-R, apoER2 or wild-type RAW264.7 macrophages were stimulated with apoE (5 μg/mL) for 24 h in the absence or presence of PP1 (10 μmol/L) or SB203580 (20 μmol/L). Incubation media were analyzed for IL1-RA concentrations. Shown are results from 4 independent experiments. § - p<0.01; # - p<0.001, apoE−inhibitor vs. apoE+inhibitor.

ApoE directs macrophages towards the M2 phenotype in vivo

To assess the influence of apoE on macrophage polarization under in vivo condition, chimeric mice were created by transplanting bone marrow (BM) from C57Bl/6 (wild type, WT) or apoE-deficient (apoE−/−) mice into apoE-deficient animals. C57Bl/6 macrophages were previously demonstrated to synthesize and secrete apoE in a paracrine fashion. As shown in Fig. 6A (left upper panel), genotyping of genomic DNA isolated from peripheral leucocytes of apoE−/−→ apoE−/− transplanted animals revealed one PCR product characteristic for apoE-deficient mice, whereas an additional WT-specific PCR product was seen in WT→apoE−/− transplanted animals. In addition, apoE was detected in supernatants of peritoneal macrophages elicited from WT→apoE−/−- but not apoE−/−→ apoE−/−-transplanted animals (Fig. 6A, left lower panel). Finally, WT→apoE−/− transplanted animals displayed significantly reduced cholesterol levels and improved plasma lipoprotein profile (Fig. 6A, right panel). Collectively, these data demonstrate a successful repopulation of BM-ablated apoE−/− mice with macrophages producing functional apoE. No significant differences were observed between apoE−/− - and WT-transplanted animals with regard to plasma levels of pro-inflammatory cytokines (not shown). However, concentrations of the anti-inflammatory IL-1RA were significantly reduced and elevated in animals, which received BM without and with apoE-secreting macrophages, respectively (Fig. 6B). To characterize the functional macrophage phenotype in transplanted animals, peritoneal macrophages were examined for the expression of M1/M2 polarization markers. As shown in Fig. 6C, macrophages obtained from WT→apoE−/−-transplanted animals were characterized by reduced production of M1 cytokine IL-12, whereas the production of M2 cytokine IL-1RA was increased. In addition, these cells demonstrated reduced surface expression of MHC-II, an M1 polarization marker (WT: 112 ± 28; apoE−/− 78 ± 24 arbU, n=6, p<0.05), whereas the expression of CD206, an M2 polarization marker was increased (WT: 106 ± 30; apoE−/− 147 ± 34 arbU, n=6, p<0.05) (Fig. 6D).

Figure 6. ApoE promotes macrophage M2 polarization in vivo.

Figure 6

Open in a new tab

ApoE-deficient mice were transplanted with bone marrow obtained from apoE−/− (BMT-E−/−, n=6) or WT (BMT-E+/+, n=6) mice as described in Methods. A. Verification of reconstitution with donor hematopoietic cells by PCR amplification of murine apoE using genomic DNA from blood cells (BC; upper left panel), immunoblot of apoE in cell media from peritoneal macrophages (PM) (lower left panel), and cholesterol distribution among serum lipoprotein fractions (right panel). Note normalization of lipoprotein profile in apoE−/− mice transplanted with WT bone marrow. B. IL-1RA concentrations in sera from C57Bl6 control mice, or apoE−/− or WT bone marrow transplanted C57Bl6 mice were determined by ELISA. C. PM supernatants from C57Bl6 control mice, C57Bl6 or mice transplanted with apoE−/− or WT bone marrow were analyzed for IL-12 and IL-1RA concentrations by ELISA. Results were normalized for cell protein content. * - p<0.05; § - p<0.01; apoE−/− vs. WT. D. MHC-II and CD206 cell surface expression was analyzed by flow cytometry in peritoneal macrophages from mice transplanted with apoE−/− or WT bone marrow. Representative histograms were superimposed for comparison. Isotype-matched irrelevant immunoglobulins were used as negative controls (Ctrl).

DISCUSSION

Previous studies support a role of apoE as an immunomodulatory agent. Initial observations demonstrated that the infection of apoE knock-out mice with Listeria monocytogenes or Klebsiella pneumonie results in an increased susceptibility to death as well as elevated serum levels of pro-inflammatory cytokines (M1) cytokines such as TNF-α as compared to wild-type animals [15,16]. Moreover, apoE-deficient mice injected with microbial stimuli (i.e. poly(I:C), LPS), which drive macrophages into the M1 inflammatory state, presented with exaggerated TNF-α, IL-6, IL-12 and IFN-γ production that could be reversed by the administration of exogenous apoE or by the adenoviral reconstitution of apoE expression in the liver [17,18]. Although these data provide evidence that apoE can suppress the pro-inflammatory response in vivo, none of the previous studies has assessed the ability of this apolipoprotein to modulate the functional polarization of macrophages. Our present study provides several pieces of evidence documenting that apoE in concentrations expected in the vasculature [19] primes macrophages into alternative M2 phenotype with anti-inflammatory properties. First, the exposure of apoE receptor-expressing macrophages to apoE led to the expression and/or the liberation of several markers (i.e. Arg-1, Fizz1/Relm, SOCS3, IL-1RA) widely recognized as attributes of M2 polarization. Second, functional characteristics of macrophages exposed to apoE including reduced migration and attenuated ROS generation and cytotoxicity as well as up-regulated phagocytic activity was congruent with a typical M2 phenotype. Third, the pretreatment of macrophages with apoE attenuated the pro-inflammatory activation in response to agents inducing “classical” M1 activation profile such as poly(I:C) and IFN-γ. Fourth, primary macrophages lacking apoE receptor (VLDL-R) displayed less pronounced M2 phenotype and enhanced response to M1 inducing agents. Studies in apoE-deficient mice additionally documented that peritoneal macrophages producing substantial amounts of apoE display decreased markers of M1 and enhanced markers of M2 phenotype. Taken together, these observations suggest that apoE acting via surface apoE receptors reprograms macrophages towards the M2 phenotype and thereby generates cell population with enhanced anti-inflammatory properties.

Monocytes/macrophages assume a critical role in the development of atherosclerosis by infiltrating the arterial wall, where they produce oxidative stress and contribute to perpetuation of pro-inflammatory processes [20,21]. Several functional properties of macrophages exposed to apoE are consistent with the notion that these cells exhibit attenuated pro-inflammatory potential. For instance, reduced motility and propensity to produce ROS would be expected to both reduce the ingress of monocytes into the intima and to locally restrict the oxidative stress and the in situ generation of modified lipoproteins. More importantly, the acquisition of M2 phenotype due to interaction with apoE confers upon macrophages the resistance to induction of NF-κB and STAT1, two transcription factors activated by pro-inflammatory agents commonly encountered in the atherosclerotic environment. Both, activated NF-κB and STAT1 have been detected within arterial lesions, where they co-localize with macrophages, and the increased activity of NF-κB has been actually observed in aortas from apoE-deficient mice [22–24]. In this context, it is worth noticing that several anti-atherogenic factors known to suppress the NF-κB and/or STAT1 activity have been demonstrated to favor M1→M2 phenotypic switching in monocytes and macrophages. For instance, agonists of peroxisome proliferation-activated receptor γ (PPARγ) - a ligand-activated nuclear receptor with potent anti-inflammatory properties, sphingosine 1-phopshate (S1P) - a lipid constituent of HDL, and FK506 – an immunosuppressive drug, were all shown to induce an anti-inflammatory activation profile in mouse and human macrophages [25–27]. In addition, enhancement of M2 functional phenotype was observed in macrophages co-cultured with CD4+CD25+ regulatory T cells - a lymphocyte subset with potent inflammation-suppressing and anti-atherogenic properties [28]. Finally, deficient CD40-TRAF6 signaling was recently shown to drive macrophages towards anti-inflammatory M2 signature and to protect against atherosclerosis in apoE-deficient mice [29]. Collectively, these observations suggest that skewing macrophage towards M2 functional phenotype may represent a universal mechanism utilized by various protective factors to counteract the development of intravascular inflammation and atherosclerosis.

The molecular mechanism underlying the immunomodulatory effects of apoE on macrophages remains unclear. As apoE absorbs cholesterol from cell membranes, it cannot be excluded that the cholesterol depletion-induced perturbation of membrane microenvironment triggers the signaling cascade required for functional polarization of macrophages. However, apoA-I – another apolipoprotein depleting membranes of cholesterol - failed to affect macrophage phenotype (Baitsch D, Nofer JR - unpublished), suggesting that the observed effects were specific for apoE. The results of the present study argue against the role of intermediate macrophage products such as IL-4 and IL-13 and suggest that the direct effect of apoE mediated via specific receptors together with the ensuing activation of the intracellular signaling machinery is required for effective switching of the functional macrophage phenotype. Both, VLDL-R and apoER2 were previously demonstrated to bind (via a NPXY tetra-aminoacid motif) and to phosphorylate an intracellular adaptor protein Dab1, which in turn mobilizes a common set of signaling modules including protein tyrosine kinase Fyn as well as phosphatidylinositol 3-kinase (PI3K) and protein kinase Akt [14,30]. However, Dab1 is expressed neither in RAW267.4 nor in primary murine macrophages (Bock HH, unpublished) and, consequently, apoE failed to activate Akt in cells overexpressing VLDL-R or apoER2. Our results suggest rather that activation of p38MAPK represents a centerpiece of intracellular signaling steering the M1→M2 phenotypic conversion. This possibility is supported by the observation that pharmacological p38MAPK inhibition fully abolished the production of M2 polarization marker IL-1RA. In this context, it is of note that in human platelets engagement of apoER2 leads to a potent activation of p38MAPK in a process dependent on the Src family kinase Fgr and blocked in the presence of Src kinase inhibitor PP1 [31]. In our hands PP1 inhibited both the apoE-induced p38MAPK activation and the IL-1RA production suggesting the involvement of a protein tyrosine kinase of as yet unknown identity. Taken together, these data point to common signaling mechanisms utilized by apoER2 (and possibly also by VLDL-R) to modulate various functional aspects of cells of hematopoietic origin.

The present data do not allow any conclusion with regard to the role played by VLDL-R or apoER2 in the pathogenesis of atherosclerosis. Various isoforms of both receptors were previously found to be expressed in the vasculature and VLDL-R appears to be particularly abundant in atherosclerotic lesions, where it co-localizes with macrophages and likely facilitates foam cell formation [32–35]. Actually, transplantation of VLDL-R+ macrophages into VLDL-R-deficient mice accelerated the development of atherosclerotic lesions [36]. However, neither generalized nor macrophage-specific VLDL-R deficiency conferred protection against atherosclerosis and even increased intimal thickening after injury has been observed in VLDL-R-deficient mice [36–38]. These observations indicate that the pro-atherogenic effect of VLDL-R arising from the enhanced cholesterol uptake by macrophages may be counterbalanced by some as yet unidentified atheroprotective effects. Basing on present results it is tempting to speculate that the anti-inflammatory macrophage polarization triggered by VLDL-R signaling mitigates pro-atherogenic effects related to VLDL-R-mediated ingestion of cholesterol-rich lipoproteins.

In conclusion, our results demonstrate that apoE signaling via VLDL-R or apoER2 promotes phenotypic switching of macrophages to a novel, anti-inflammatory phenotype. These macrophages show decreased activation of transcription factors NF-κB and STAT1 and reduced motility and production of free radicals and pro-inflammatory cytokines. Macrophage conversion from pro-inflammatory M1 to anti-inflammatory M2 phenotype represents a novel anti-atherogenic activity of apoE and may be part of a universal mechanism utilized by various protective agents to counteract the development of atherosclerotic lesions.

Supplementary Material

Acknowledgments

SOURCES OF FUNDING

This work was supported by a grant NO110441 from the Innovative Medizinische Forschung (IMF) and intramural resources of the Center for Laboratory Medicine to J.-R. N. and grant EC116/3-6 from the Deutsche Forschungsgemeinschaft (DFG) to A.v E (administered by J.-R. N.).

The expert technical assistance of Alois Röttrige, Cornelia Richter-Elzenheimer, Beate Schulte, Katrin Tkotz, Jonathan Göldner, and Bertram Tambyrajah is gratefully acknowledged.

Footnotes

DISCLOSURES

None

References

  • 1.Greenow K, Pearce NJ, Ramji DP. The key role of apolipoprotein E in atherosclerosis. J Mol Med. 2005;83:329–42. doi: 10.1007/s00109-004-0631-3. [DOI] [PubMed] [Google Scholar]
  • 2.Linton MF, Atkinson JB, Fazio S. Prevention of atherosclerosis in apolipoprotein E-deficient mice by bone marrow transplantation. Science. 1995;267:1034–1037. doi: 10.1126/science.7863332. [DOI] [PubMed] [Google Scholar]
  • 3.Boisvert WA, Spangenberg J, Curtiss LK. Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by bone marrow transplantation. J Clin Invest. 1995;96:1118–1124. doi: 10.1172/JCI118098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Fazio S, Babaev VR, Murray AB, Hasty AH, Carter KJ, Gleaves LA, Atkinson JB, Linton MF. Increased atherosclerosis in mice reconstituted with apolipoprotein E null macrophages. Proc Natl Acad Sci U S A. 1997;94:4647–4652. doi: 10.1073/pnas.94.9.4647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Mosser D. The many faces of macrophage activation. J Leukoc Biol. 2003;73:209–12. doi: 10.1189/jlb.0602325. [DOI] [PubMed] [Google Scholar]
  • 6.Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol. 2004;25:677–86. doi: 10.1016/j.it.2004.09.015. [DOI] [PubMed] [Google Scholar]
  • 7.Martinez FO, Sica A, Mantovani A, Locati M. Macrophage activation and polarization. Front Biosci. 2008;13:453–61. doi: 10.2741/2692. [DOI] [PubMed] [Google Scholar]
  • 8.Geissmann F, Gordon S, Hume DA, Mowat AM, Randolph GJ. Unravelling mononuclear phagocyte heterogeneity. Nat Rev Immunol. 2010;10:453–60. doi: 10.1038/nri2784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Khallou-Laschet J, Varthaman A, Fornasa G, Compain C, Gaston AT, Clement M, Dussiot M, Levillain O, Graff-Dubois S, Nicoletti A, Caligiuri G. Macrophage plasticity in experimental atherosclerosis. PLoS One. 2010;5:e8852. doi: 10.1371/journal.pone.0008852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Trommsdorff M, Gotthardt M, Hiesberger T, Shelton J, Stockinger W, Nimpf J, Hammer RE, Richardson JA, Herz J. Reeler/Disabled-like disruption of neuronal migration in knockout mice lacking the VLDL receptor and ApoE receptor 2. Cell. 1999;97:689–701. doi: 10.1016/s0092-8674(00)80782-5. [DOI] [PubMed] [Google Scholar]
  • 11.Frykman PK, Brown MS, Yamamoto T, Goldstein JL, Herz J. Normal plasma lipoproteins and fertility in gene-targeted mice homozygous for a disruption in the gene encoding very low density lipoprotein receptor. Proc Natl Acad Sci U S A. 1995;92:8453–7. doi: 10.1073/pnas.92.18.8453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Donaldson DD, Whitters MJ, Fitz LJ, Neben TY, Finnerty H, Henderson SL, O’Hara RM, Jr, Beier DR, Turner KJ, Wood CR, Collins M. The murine IL-13 receptor alpha 2: molecular cloning, characterization, and comparison with murine IL-13 receptor alpha 1. J Immunol. 1998;161:2317–24. [PubMed] [Google Scholar]
  • 13.Deepak P, Kumar S, Acharya A. Interleukin-13 neutralization modulates interleukin-13 induced suppression of reactive oxygen species production in peritoneal macrophages in a murine T-cell lymphoma. Cell Immunol. 2008;251:72–7. doi: 10.1016/j.cellimm.2008.03.005. [DOI] [PubMed] [Google Scholar]
  • 14.Schneider W, Nimpf J. LDL receptor relatives at the crossroad of endocytosis and signaling. Cell Mol Life Sci. 2003;60:892–903. doi: 10.1007/s00018-003-2183-Z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Roselaar SE, Daugherty A. Apolipoprotein E-deficient mice have impaired innate immune responses to Listeria monocytogenes in vivo. J Lipid Res. 1998;39:1740–3. [PubMed] [Google Scholar]
  • 16.de Bont N, Netea MG, Demacker PN, Kullberg BJ, van der Meer JW, Stalenhoef AF. Apolipoprotein E-deficient mice have an impaired immune response to Klebsiella pneumoniae. Eur J Clin Invest. 2000;30:818–22. doi: 10.1046/j.1365-2362.2000.00715.x. [DOI] [PubMed] [Google Scholar]
  • 17.Van Oosten M, Rensen PC, Van Amersfoort ES, Van Eck M, Van Dam AM, Breve JJ, Vogel T, Panet A, Van Berkel TJ, Kuiper J. Apolipoprotein E protects against bacterial lipopolysaccharide-induced lethality. A new therapeutic approach to treat gram-negative sepsis. J Biol Chem. 2001;276:8820–4. doi: 10.1074/jbc.M009915200. [DOI] [PubMed] [Google Scholar]
  • 18.Ali K, Middleton M, Puré E, Rader DJ. Apolipoprotein E suppresses the type I inflammatory response in vivo. Circ Res. 2005;97:922–7. doi: 10.1161/01.RES.0000187467.67684.43. [DOI] [PubMed] [Google Scholar]
  • 19.Sloop CH, Dory L, Roheim PS. Interstitial fluid lipoproteins. J Lipid Res. 1987;28:225–237. [PubMed] [Google Scholar]
  • 20.von der Thüsen JH, Kuiper J, van Berkel TJ, Biessen EA. Interleukins in atherosclerosis: molecular pathways and therapeutic potential. Pharmacol Rev. 2003;55:133–66. doi: 10.1124/pr.55.1.5. [DOI] [PubMed] [Google Scholar]
  • 21.Weber C, Zernecke A, Libby P. The multifaceted contributions of leukocyte subsets to atherosclerosis: lessons from mouse models. Nat Rev Immunol. 2008;8:802–15. doi: 10.1038/nri2415. [DOI] [PubMed] [Google Scholar]
  • 22.Brand K, Page S, Rogler G, Bartsch A, Brandl R, Knuechel R, Page M, Kaltschmidt C, Baeuerle PA, Neumeier D. Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion. J Clin Invest. 1996;97:1715–22. doi: 10.1172/JCI118598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Lim WS, Timmins JM, Seimon TA, Sadler A, Kolodgie FD, Virmani R, Tabas I. Signal transducer and activator of transcription-1 is critical for apoptosis in macrophages subjected to endoplasmic reticulum stress in vitro and in advanced atherosclerotic lesions in vivo. Circulation. 2008;117:940–51. doi: 10.1161/CIRCULATIONAHA.107.711275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Hishikawa K, Nakaki T, Fujita T. Oral flavonoid supplementation attenuates atherosclerosis development in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2005;25:442–6. doi: 10.1161/01.ATV.0000148404.24271.fc. [DOI] [PubMed] [Google Scholar]
  • 25.Bouhlel MA, Derudas B, Rigamonti E, Dièvart R, Brozek J, Haulon S, Zawadzki C, Jude B, Torpier G, Marx N, Staels B, Chinetti-Gbaguidi G. PPARgamma activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties. Cell Metab. 2007;6:137–43. doi: 10.1016/j.cmet.2007.06.010. [DOI] [PubMed] [Google Scholar]
  • 26.Hughes JE, Srinivasan S, Lynch KR, Proia RL, Ferdek P, Hedrick CC. Sphingosine-1-phosphate induces an antiinflammatory phenotype in macrophages. Circ Res. 2008;102:950–8. doi: 10.1161/CIRCRESAHA.107.170779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Bai L, Gabriels K, Wijnands E, Rousch M, Daemen MJ, Tervaert JW, Biessen EA, Heeneman S. Low- but not high-dose FK506 treatment confers atheroprotection due to alternative macrophage activation and unaffected cholesterol levels. Thromb Haemost. 2010;104:143–50. doi: 10.1160/TH09-07-0502. [DOI] [PubMed] [Google Scholar]
  • 28.Tiemessen MM, Jagger AL, Evans HG, van Herwijnen MJ, John S, Taams LS. CD4+CD25+Foxp3+ regulatory T cells induce alternative activation of human monocytes/macrophages. Proc Natl Acad Sci U S A. 2007;104:19446–51. doi: 10.1073/pnas.0706832104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Lutgens E, Lievens D, Beckers L, Wijnands E, Soehnlein O, Zernecke A, Seijkens T, Engel D, Cleutjens J, Keller AM, Naik SH, Boon L, Oufella HA, Mallat Z, Ahonen CL, Noelle RJ, de Winther MP, Daemen MJ, Biessen EA, Weber C. J Exp Med. 2010;207:391–404. doi: 10.1084/jem.20091293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.May P, Herz J, Bock HH. Molecular mechanisms of lipoprotein receptor signalling. Cell Mol Life Sci. 2005;62:2325–38. doi: 10.1007/s00018-005-5231-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Korporaal SJ, Relou IA, van Eck M, Strasser V, Bezemer M, Gorter G, van Berkel TJ, Nimpf J, Akkerman JW, Lenting PJ. Binding of low density lipoprotein to platelet apolipoprotein E receptor 2′ results in phosphorylation of p38MAPK. J Biol Chem. 2004;279:52526–34. doi: 10.1074/jbc.M407407200. [DOI] [PubMed] [Google Scholar]
  • 32.Korschineck I, Ziegler S, Breuss J, Lang I, Lorenz M, Kaun C, Ambros PF, Binder BR. Identification of a novel exon in apolipoprotein E receptor 2 leading to alternatively spliced mRNAs found in cells of the vascular wall but not in neuronal tissue. J Biol Chem. 2001;276:13192–7. doi: 10.1074/jbc.M011795200. [DOI] [PubMed] [Google Scholar]
  • 33.Multhaupt HA, Gåfvels ME, Kariko K, Jin H, Arenas-Elliot C, Goldman BI, Strauss JF, 3rd, Angelin B, Warhol MJ, McCrae KR. Expression of very low density lipoprotein receptor in the vascular wall. Analysis of human tissues by in situ hybridization and immunohistochemistry. Am J Pathol. 1996;148:1985–97. [PMC free article] [PubMed] [Google Scholar]
  • 34.Nakazato K, Ishibashi T, Shindo J, Shiomi M, Maruyama Y, Nakazato K, Ishibashi T, Shindo J, Shiomi M, Maruyama Y. Expression of very low density lipoprotein receptor mRNA in rabbit atherosclerotic lesions. Am J Pathol. 1996;149:1831–8. [PMC free article] [PubMed] [Google Scholar]
  • 35.Hiltunen TP, Luoma JS, Nikkari T, Ylä-Herttuala S. Expression of LDL receptor, VLDL receptor, LDL receptor-related protein, and scavenger receptor in rabbit atherosclerotic lesions: marked induction of scavenger receptor and VLDL receptor expression during lesion development. Circulation. 1998;97:1079–86. doi: 10.1161/01.cir.97.11.1079. [DOI] [PubMed] [Google Scholar]
  • 36.Eck MV, Oost J, Goudriaan JR, Hoekstra M, Hildebrand RB, Bos IS, van Dijk KW, Van Berkel TJ. Role of the macrophage very-low-density lipoprotein receptor in atherosclerotic lesion development. Atherosclerosis. 2005;183:230–7. doi: 10.1016/j.atherosclerosis.2005.03.045. [DOI] [PubMed] [Google Scholar]
  • 37.Yagyu H, Lutz EP, Kako Y, Marks S, Hu Y, Choi SY, Bensadoun A, Goldberg IJ. Very low density lipoprotein (VLDL) receptor-deficient mice have reduced lipoprotein lipase activity. Possible causes of hypertriglyceridemia and reduced body mass with VLDL receptor deficiency. J Biol Chem. 2002;277:10037–43. doi: 10.1074/jbc.M109966200. [DOI] [PubMed] [Google Scholar]
  • 38.Tacken PJ, Delsing DJ, Gijbels MJ, Quax PH, Havekes LM, Hofker MH, van Dijk KW. VLDL receptor deficiency enhances intimal thickening after vascular injury but does not affect atherosclerotic lesion area. Atherosclerosis. 2002;162:103–10. doi: 10.1016/s0021-9150(01)00697-9. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS425470-supplement-supplement_1.pdf (632.6KB, pdf)

RESOURCES