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The Journal of Immunology, 2004, 173: 6366-6375.
Copyright © 2004 by The American Association of Immunologists

Apolipoprotein E Modulates Clearance of Apoptotic Bodies In Vitro and In Vivo, Resulting in a Systemic Proinflammatory State in Apolipoprotein E-Deficient Mice1

David J. Grainger2,*, Jill Reckless* and Elaine McKilligin{dagger}

* Department of Medicine, Addenbrooke’s Hospital, University of Cambridge, Cambridge, United Kingdom; and {dagger} Atherosclerosis Department, Cardiovascular and Urology CEDD, GlaxoSmithKline, Stevenage, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materils and Methods
 Results
 Discussion
 References
 
Apolipoprotein E (apoE) is a 34-kDa glycoprotein involved in lipoprotein transport through interaction with the low-density lipoprotein receptor and related receptors. Recently, it has become clear that apoE binding to its receptors plays a role both in development and in control of the immune system. In this study, we show that apoE modulates the rate of uptake of apoptotic cells by macrophages. In vitro, apoE-deficient macrophages ingest less apoptotic thymocytes (but not latex beads) than wild-type macrophages, and this defect can be corrected by addition of exogenous apoE protein. In vivo, the number of dying macrophages is increased in a range of tissues, including lung and brain. Possibly in response to the larger numbers of persistent apoptotic bodies, the number of live macrophages in these tissues are also increased compared with those of wild-type control mice. In addition to the significant changes in macrophage population dynamics we observed, levels of the proinflammatory cytokine TNF-{alpha} and the positive acute phase reactant fibrinogen are also elevated in the livers from apoE-deficient mice. In contrast, neither deletion of the gene encoding the LDL receptor nor cholesterol feeding of wild-type mice affected either the number of apoptotic bodies or the number of live macrophages. We conclude that apoE deficiency results in impaired clearance of apoptotic cell remnants and a functionally relevant systemic proinflammatory condition in mice, independent of its role in lipoprotein metabolism. Any similar reduction of apoE activity in humans may contribute to the pathogenesis of a wide range of chronic diseases including atherosclerosis, dementia, and osteoporosis.


   Introduction
Top
 Abstract
 Introduction
Materils and Methods
 Results
 Discussion
 References
 
Apolipoprotein E (apoE),3 in combination with apolipoprotein B, constitutes the majority of the protein present in the triglyceride-rich lipoprotein particles low-density lipoprotein (LDL) and very low LDL (VLDL). The apoE component is a ligand for the LDL receptor (LDLR) and a family of LDLR-related proteins or LRPs (1). Consequently, homozygous deletion of the apoE gene in mice has a dramatic effect on cholesterol transport, resulting in a large increase in the plasma levels of LDL and VLDL due to the failure of LDLR and LRP-mediated clearance of these lipoproteins from the blood (2, 3). The most obvious phenotype of apoE-deficient mice is the development of vascular lipid lesions resembling early human atherosclerosis, even on a diet with normal fat content (2, 3). It has generally been assumed that defective lipoprotein metabolism is the major cause of the development of vascular lipid lesions in apoE-deficient mice. However, the observation that macrophage-specific reintroduction of apoE into apoE-deficient mice ameliorates lipid lesion formation independent of any effects on systemic lipoprotein levels (4) suggests that apoE may be exerting local effects at the blood vessel wall as well as effects on systemic lipoprotein trafficking, predominantly by the liver.

It is plausible that apoE plays a similar role in humans as in mice: three common allelic variants of the apoE gene exist, designated apo{epsilon}2, {epsilon}3, and {epsilon}4 encoding apoE2, E3, and E4, respectively. ApoE2 and E4 differ from the most common E3 isotype by a single amino acid substitution in each case. It is now clear that the presence of even a single copy of the apo{epsilon}4 allele results in an increased risk of coronary heart disease compared with the wild-type (WT) apo{epsilon}3 homozygote (5, 6). However, the impact of the apo{epsilon}2 allele is less clear: it is associated with complex defects in lipoprotein metabolism such as type III hyperlipoproteinemia and has been associated with both an increase and a decrease in atherosclerosis, depending on the study design (6). The precise molecular basis for the association between apoE genotype and cardiovascular disease incidence is uncertain, but it is possible that the isotypes differ in the ability to mediate plasma clearance of lipoprotein particles.

However, genetic studies of the apoE haplotypes in a range of different diseases have suggested a much broader role for apoE (7). The presence of the apo{epsilon}4 allele has been associated with increased risk of Alzheimer’s disease (7, 8, 9) and possibly also osteoporosis (10, 11). Indeed, the associations between apoE haplotype and a range of prevalent disorders are sufficiently strong that apoE remains one of the few loci which has been unequivocally associated with life span: the apo{epsilon}2 allele is overrepresented among octogenarians, suggesting that the apoE genotype contributes to longevity (12).

Subtle variations in lipoprotein transport in the brain might contribute to the pathogenesis of Alzheimer’s disease, but it is presently difficult to rationalize any association between apoE genotype and bone mineral density on the basis of this mechanism. Alternatively, apoE may have a role, independent of its function in lipoprotein metabolism, in a process which is important to the pathogenesis of Alzheimer’s disease, atherosclerosis, and osteoporosis (1, 7).

Recent studies have suggested that the LRP family of receptors may transduce a signal important in regulating cell migration during development, possibly as a component of the Wnt receptor complex (1, 7, 13, 14). Furthermore, apoE may regulate local inflammation through receptor-mediated signaling cascades independent of lipoprotein transport. For example, apoE, as well as a 14-aa receptor-binding peptide that does not bind cholesterol, was able to suppress the local inflammatory response to experimental cerebral ischemia in vivo (15, 16). Cell culture studies suggest this effect may be mediated through suppression of macrophage function (17, 18). Studies of chronic infections in apoE-deficient mice lead to a similar conclusion that apoE somehow suppresses macrophage function (19, 20, 21, 22).

Misregulation of macrophage population dynamics could plausibly be a key pathogenic mechanism in a range of prevalent diseases (23), including Alzheimer’s disease (24, 25, 26), atherosclerosis (27, 28), and osteoporosis (29). Activation of the microglia, the cerebral endogenous tissue macrophage, is a component of senile plaque formation in Alzheimer’s disease (24). Similarly, excess activity of the osteoclast (a macrophage-like cell in bone which is derived from the circulating monocyte pool) contributes to the development of osteoporosis (29). Inappropriate macrophage accumulation also participates in the development of atherosclerosis (27, 28), and, in particular, results in destabilization of the atherosclerotic plaque through secretion of matrix-degrading enzymes (28).

In this study, we have tested the hypothesis that apoE modulates the uptake of apoptotic cells by macrophages, and consequently regulates macrophage population dynamics in multiple tissues by a mechanism independent of lipoprotein transport. In addition to studying the effects of apoE on macrophage phagocytosis in vitro, we have examined the macrophage population in a range of tissues in vivo, including liver, brain, and lung, in mice deficient in apoE.


   Materils and Methods
Top
 Abstract
 Introduction
 Materils and Methods
Results
 Discussion
 References
 
In vitro phagocytosis assays

Murine peritoneal macrophages were used for all phagocytosis assays. Macrophages were aseptically isolated from either C57BL/6 (WT) or apoE–/– mice by washing out the mouse peritoneum with ice-cold sterile HBSS (Sigma-Aldrich, St. Louis, MO). The peritoneal exudate cells were stored in precooled sterile glass tubes and were then washed twice with RPMI 1640 medium (pelleted at 300 xg for 10 min). The cell pellet was finally resuspended in RPMI 1640 plus 10% FCS and plated at 5 x 105 cells/ml onto plastic tissue culture chamber slides (Nunc, Roskilde, Denmark). The macrophages were allowed to adhere for 2 h at 37°C, then washed with cold Dulbecco’s PBS before incubation in RPMI 1640 plus 10% FCS until used in the phagocytosis assay between 4 and 10 days after isolation. All cells for use in phagocytosis assays were washed thoroughly and incubated under serum-free conditions to eliminate any possible effects due to the presence of bovine apoE in the FCS used for cell maintenance.

The latex bead phagocytosis assay was adapted from Ichinose et al. (30). Briefly, 2-µm fluorescent microspheres (carboxylate modified; Molecular Probes, Eugene, OR) in distilled water at 4.5 x 109 beads/ml were coated with 1% BSA and then sonicated for 5 min. Macrophage monolayers were washed with HEPES-buffered saline (pH 7.5) and preincubated for 30 min with 250 µl of the same solution. The BSA-coated latex beads were added to give 2.5 x 106 beads/well and then the macrophages were incubated for 1 h at 37°C, during which phagocytosis occurred. Uningested beads were then removed by five vigorous washes with ice-cold Dulbecco’s PBS. Macrophages were then released by addition of 0.25% trypsin (type IIS from porcine pancreas; Sigma-Aldrich) for 2 h at 37°C and fixed by addition of glutaraldehyde (0.5% final concentration). The number of ingested particles was measured by flow cytometry using a FACStar (BD Biosciences, Mountain View, CA) analyzing 10,000 cells/tube and the average number of particles ingested per macrophage (the phagocytic index) was calculated using FCSPress software (Cambridge, U.K.).

The apoptotic thymocyte phagocytosis assay was adapted from the procedures described previously (30, 31). Thymocytes were prepared from C57BL/6 or ApoE–/– mice by removing the thymus into RPMI 1640 plus 10% FCS buffered with 20 mM Tris-HCl (pH 7.2) and passing it through a cell dissociation sieve with 40-mesh screen (Sigma-Aldrich). The resultant cell suspension was pelleted (500 x g for 5 min) and resuspended in RPMI 1640 plus 10% FCS at 107 cells/ml. The day before the cells were used in a phagocytosis assay (3–9 days after putting them into culture), the thymocytes were washed three times with sterile Dulbecco’s PBS and reconstituted to 5 x 106 cells/ml and then labeled with Cell Tracker Green (2 µM final concentration; Molecular Probes) for 30 min at 37°C (31). The labeled cells were then washed in PBS and incubated in fresh serum-free RPMI 1640 medium for 30 min at 37°C, washed again in PBS, and then incubated in serum-free RPMI 1640 overnight. Removal of serum yielded >70% apoptotic thymocytes as measured by annexin V staining and flow cytometry (data not shown). Phagocytosis assays were performed as for latex beads (30), except that 2 x 106 apoptotic thymocytes per well were added. Ingested thymocytes were counted by fixing the macrophages on the glass slides with 1% acetic acid in 70% ethanol for 90 min and examining them under a fluorescence microscope (Provis AX; Olympus, Melville, NY) attached to an image analysis system. The number of ingested thymocytes and the total of number of macrophages were counted in each of 18 fields of view per well and the average phagocytic index was calculated.

Tissue preparation

Adult male mice (either C57BL/6, ApoE–/– (3), or LDLR–/– mice (32); six animals per group) were sacrificed by CO2 asphyxiation and blood was drawn by cardiac puncture for preparation of serum as previously described (33). Tissues (left lobe of liver, left lung, and left hemisphere of brain) were rapidly dissected out into ice-cold saline, then embedded in OCT embedding medium and frozen at –80°C. For the animals receiving a dietary lipid challenge, normal chow was replaced with a high lipid content chow (1.25% cholesterol, 7.5% saturated fat) for 10 wk before sacrifice. All other animals received normal chow diet and water ad libitum throughout.

Cryosections (4 µm) were then prepared from each tissue and collected onto poly-L-lysine-coated slides and fixed in ice-cold acetone for 90 s, air dried, and frozen at –20°C until analyzed. Transverse sections were taken from a point approximately in the middle of the tissue on the anterioposterior axis, over a distance of 4 mm. For each Ag (or group of Ags in double- or triple-color immunofluorescence experiments), 16 sections spaced evenly over 4 mm (every 250 µm) were used, with 10 sections receiving primary Ab and 6 sections (selected randomly from the 16) serving as controls with the first Ab omitted, as recommended by Mosedale et al. (34).

Immunofluorescence staining

All immunofluorescence staining was performed as described by Mosedale et al. (34) under the conditions optimized for quantitative immunofluorescence. Briefly, sections were rehydrated in PBS containing 3% fatty acid-free BSA for 30 min, then exposed to primary Ab in PBS/3% BSA for 18 h. After 3 x 3-min washes in PBS, slides were exposed to the appropriate fluorescently labeled secondary Ab at 25 µg/ml in PBS/3% BSA for 6 h, except where directly labeled primaries were used. After three additional washes in PBS, slides were rinsed in water, air dried, and mounted under Citifluor AF-1 and then stored at –20°C until analyzed using the image analysis system as previously described (34). The following primary Abs were used: anti-macrophage F4/80 Ag (MCAP497, 25 µg/ml; Serotec, Oxford, U.K.), anti-CD11b (M1/70 (MCA74G), 25 µg/ml; Serotec), anti-MHC class II (I-Ab) (MCA1500F, 20 µg/ml; Serotec), anti-fibrinogen (4440-8004, 20 µg/ml; Biogenesis; Poole, U.K.), anti-murine TNF-{alpha} (AB-410-NA, 50 µg/ml; R&D Systems, Minneapolis, MN), and anti-proliferating cell nuclear Ag (M0879, 12 µg/ml; DakoCytomation, Carpinteria, CA). All secondary Abs were minimum cross-reactivity donkey Abs (Jackson ImmunoResearch Laboratories, West Grove, PA), All Ab solutions also contained Hoechst 33342 (1 µg/ml final concentration) to counterstain nuclei, visible on the blue channel.

Detection of apoptotic and necrotic cells

Dying cells were detected using the TUNEL reaction, as previously described (35), using the In Situ Cell Death Detection kit (Roche, Basel, Switzerland) in accordance with the manufacturer’s instructions. Sections pretreated with bovine DNase I (10 µg/ml final concentration in 50 mM Tris (pH 7.5) and 1 mM MgCl2; Roche) were used as positive controls; omission of the fluorescein-labeled dUTP was used as the negative control. In mouse liver, ~70% of the TUNEL+ cells also stained positively for activated caspase-3 (using the CM-1 Ab) and showed signs of nuclear condensation when viewed for Hoechst 33342 fluorescence, suggesting the majority of the cells detected as TUNEL+ using this kit were undergoing apoptosis, as opposed to necrosis, according to the definitions of Stadelmann and Lassmann (36), as claimed by the manufacturers.

Lipoprotein profile analysis

Pooled serum samples were subjected to gel filtration chromatography using a Sepharose 6B column exactly as previously described (37). Cholesterol was detected in the resulting fractions using the cholesterol oxidase method and assigned to the various lipoprotein classes on the basis of apolipoprotein elution profiles analyzed by gel electrophoresis and Western blotting as previously described (38).


   Results
Top
 Abstract
 Introduction
 Materils and Methods
 Results
Discussion
 References
 
Effect of apoE deficiency on macrophage uptake of apoptotic cells in vitro

Peritoneal macrophages were prepared from apoE-deficient mice (E–/–) and WT mice as controls. After 96 h in culture, the E–/– and WT cells were each presented with fluorescently labeled apoptotic WT thymocytes which they ingested over a period of 1 h at 37°C. The number of thymocytes ingested by each macrophage was quantitated by immunofluorescence microscopy (Fig. 1A) as a measure of the capacity of the macrophages to clear apoptotic bodies. In this period, WT macrophages each ingested 1.65 ± 0.12 apoptotic WT thymocytes, but no ingestion was seen if live thymocytes were used (data not shown), confirming that this assay was specific for uptake of apoptotic bodies. In contrast, the E–/– macrophages ingested 25% fewer apoptotic thymocytes in the same period (p < 0.05, Student’s unpaired t test; Fig. 1B).



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  		FIGURE 1. Effect of apoE deficiency on phagocytosis in vitro. A, Fluorescence micrographs of macrophages after taking up labeled apoptotic thymocytes for 30 min at 37°C. In the control image no thymocytes were added. The images were captured at high gain so that the unlabeled macrophages are visible by autofluorescence in addition to the labeled thymocytes. Scale bar, 25 µm. B, Quantitation of the number of WT thymocytes taken up per macrophage from images such as those presented in A. The total number of thymocytes in 10 fields of view were divided by the total number of macrophages. Values are the mean ± SEM from triplicate wells in each of three experiments. C, Quantitation of the number of apoE-deficient thymocytes taken up per macrophage.The effect of preincubation with 1 µM soluble recombinant human apoE3 is also shown. D, A typical flow cytometric histogram of macrophages after uptake of fluorescently labeled latex beads for 30 min at 37°C. Cells that had taken up no beads have a low fluorescence intensity (FL1-H), while cells taking up one to three or more beads are progressively shifted to higher fluorescence intensities. A typical histogram for WT macrophages is shown in black and for apoE-deficient macrophages in gray. E, Quantitation of the number of latex beads taken up per macrophage from flow cytometric histograms such as those presented in D. Values are the mean ± SEM for triplicate wells in each of three separate experiments.

 
Flow cytometry using a FITC-labeled Ab against murine apoE demonstrated that thymocytes express apoE, albeit at a lower level than macrophages. We therefore repeated the experiment using E–/– thymocytes. Interestingly, WT macrophages did not ingest E–/– thymocytes as efficiently as they did WT thymocytes (p < 0.05, Student’s t test; Fig. 1C), and an even greater impairment in uptake of apoptotic bodies was seen when both the ingesting macrophages and the apoptotic thymocytes were apoE deficient, with ~60% fewer apoptotic cells ingested than in the WT system (p < 0.05, ANOVA; Fig. 1C). Taken together, these results demonstrate that although ingestion of apoptotic bodies can occur in the complete absence of apoE, the process is markedly attenuated.

Since ingestion of apoptotic bodies is decreased to a similar extent by deficiency of apoE on the macrophage or thymocyte partner, we tested whether exogenous addition of soluble apoE could restore normal function. Addition of human apoE3 at 1 µM (a similar concentration to human plasma (39)) reversed the attenuation of thymocyte ingestion caused by endogenous apoE deficiency (p < 0.05 vs no addition of apoE; p = 0.98 vs WT cells, Student’s unpaired t test; Fig. 1C). We conclude that apoE is an important modulator of apoptotic body uptake but it is unlikely to function in a receptor:ligand pair participating in direct cellular interaction during uptake, but to have a more complex regulatory role possibly involving signaling at the cell surface.

Next we tested whether apoE is a broad-spectrum modulator of macrophage phagocytosis or whether it has specificity for the uptake of apoptotic cells, since the molecular pathways involved in the uptake of apoptotic thymocytes vs latex beads are already known to be different (31). The uptake of fluorescently labeled latex beads, measured using flow cytometry (Fig. 1D), by E–/– macrophages was indistinguishable from that of WT macrophages (p = 0.86, Student’s unpaired t test; Fig. 1E), demonstrating that the effect of apoE on phagocytosis was relatively specific for the uptake of apoptotic bodies.

Effect of apoE deficiency on clearance of apoptotic bodies in vivo

Since apoE modulates uptake of apoptotic bodies by macrophages in vitro, we analyzed the number of apoptotic bodies present in vivo in apoE-deficient mice and WT littermate controls. Cryosections were prepared at 250-µm intervals through 4 mm of the left lobe of the liver, and dying cells and cell remnants were detected using the TUNEL reaction, which labels DNA breaks. Costaining for the general macrophage marker F4/80 revealed that the majority (>98%) of TUNEL-positive cells in both WT- and apoE-deficient liver were F4/80+ (most of the TUNEL-positive cells were macrophages). It is important to note, however, that TUNEL labeling does not only stain apoptotic cells and may stain necrotic cells or even healthy cells depending on the method of tissue preparation (36). However, in our sections many of the TUNEL-positive cells had characteristics of apoptotic cells when viewed for Hoechst fluorescence (i.e., chromatin condensation and vacuolation of the nuclei were evident). Furthermore, 60–70% of the TUNEL-positive cells also stained for active caspase-3 (data not shown). Taken together, these observations suggest that TUNEL staining provides a useful, although imperfect, index of macrophage cell death in the liver, consistent with the findings of Stadelmann and Lassmann (36). Although careful histological examination of the slides confirmed that the majority of TUNEL+F4/80+ cells were likely to be dying macrophages, we cannot exclude the possibility that a minority of such cells (up to 20% of them) may be live macrophages containing apoptotic bodies.

The number of dying macrophages (F4/80+TUNEL+) in the liver was dramatically increased in the apoE-deficient mice compared with the WT littermate controls (Fig. 2, A–C). Both the absolute number of apoptotic macrophages (p < 0.01, Student’s unpaired t test; Fig. 2B) and the proportion of the macrophage population which were TUNEL+ (p < 0.05, Student’s unpaired t test; Fig. 2C) were increased. In WT animals ~10% of the F4/80+ cells were also TUNEL+, whereas in apoE-deficient animals >50% were TUNEL+. We also examined the impact of apoE deficiency on the number of apoptotic hepatocytes (F4/80–TUNEL+ cells). However, the rate of cell death among the non-macrophage cellular compartments was very much lower than among the macrophage compartment (<2% of all of the TUNEL+ cells were F4/80–); therefore, our experiment was underpowered to detect any impact of apoE deficiency on this population (Fig. 2D).



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  		FIGURE 2. TUNEL staining of mouse liver. A, Two-color fluorescence micrographs of the same field of view from a typical section of liver from an apoE-deficient mouse. Macrophages (mostly Kupffer cells) are illuminated in the red channel (right panel) using the mAb F4/80. Cell remnants and dying cells were stained using the TUNEL reaction in the green channel (left panel). All of the TUNEL+ cells in this image are also F4/80+, but ~50% of the F4/80+ cells are TUNEL– (arrows). We cannot exclude the possibility that a minority of these TUNEL+F4/80+ cells are live macrophages containing apoptotic bodies. Scale bar, 25 µm. B, Quantification of the absolute number of TUNEL+F4/80+ cells in 10 sections of liver from each of six mice of each genotype expressed as a percentage of the total number of nuclei analyzed. C, The number of TUNEL+F4/80+ cells expressed as a percentage of the number of F4/80+ cells in the same sections analyzed in B. D, The number of TUNEL+F4/80– cells (i.e., cell remnants or dying cells which are not macrophages) expressed as a percentage of the total number of nuclei analyzed. B–D, The values are mean ± SEM for six animals.

 
The observed increase in apoptotic macrophages in the liver of apoE-deficient mice could have resulted from either an increased rate of macrophage death or a decreased rate of apoptotic body clearance. To distinguish these two possibilities, we analyzed the macrophage population more fully to provide a picture of macrophage population dynamics in mouse liver. First, we estimated the size of the liver macrophage population by expressing the total number of F4/80+ cells as a percentage of the total number of nuclei in the liver (Fig. 3, A and B). Despite the dramatic increase in the number and proportion of TUNEL+ macrophages in apoE-deficient mice, the total liver macrophage population was significantly larger in the apoE-deficient animals (p < 0.05, Student’s unpaired t test; Fig. 3B). There are at least two simple explanations for this apparent paradox: either the clearance of apoptotic bodies is significantly less efficient in apoE-deficient mice, consistent with our observations in vitro, or macrophage recruitment has been increased in the absence of apoE to an even greater extent than any increase in macrophage death.



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  		FIGURE 3. Macrophage population dynamics in mouse liver. A, Low-power fluorescence micrographs of typical liver sections from WT and apoE-deficient mice stained using the F4/80 mAb. At this magnification (scale bar, 25 µm), the macrophages appear as white specks, which are confirmed to be specific cellular staining under high magnification (inset; scale bar, 10 µm). B, Quantification of the number of F4/80+ cells in 10 sections of liver from each of six groups of mice of each genotype expressed as a percentage of the total number of nuclei analyzed. C, Quantification of the number of M1/70+ cells (expressing high levels of the integrin CD11b, representing recently recruited macrophages) in the same sections as B. The absolute number of M1/70+ cells as percentage of the total number of nuclei analyzed is depicted, and the numbers above each bar represent the number of M1/70+ cells as a fraction of the number of F4/80+ cells in the same sections. B and C, Values represent the mean ± SEM for six mice. D, A summary of macrophage population dynamics in WT (left chart) and apoE-deficient (right chart) mice. The area of each chart is proportional to the total number of F4/80+ cells, and it is subdivided to show the proportions of this macrophage population which are newly recruited (M1/70+TUNEL–), mature (M1/70–TUNEL–), or dying (M1/70–TUNEL+). The number of live, mature macrophages (M1/70–F4/80+TUNEL– cells), expressed as a percentage of the total number of nuclei, is also shown.

 
To investigate this second possibility, we exploited the observation that the integrin CD11b (detected by the mAb M1/70), which is highly expressed on monocytes, is down-regulated following recruitment into the tissue macrophage population (40, 41). As a result, the number of M1/70+F/480+ cells in the liver can be used as a surrogate index of macrophage recruitment. ApoE deficiency increased the absolute number of M1/70+F4/80+ macrophages in the liver by >2-fold (p < 0.05, Student’s unpaired t test; Fig. 3C), but this only represents a small increase in the proportion of macrophages which are newly recruited (10.0% in apoE-deficient mice compared with 8.3% in C57BL/6 mice). Thus, any increase in the rate of macrophage recruitment is substantially less than the large increase seen in the number of dead or dying macrophages. Since proliferation of the macrophage population is a very rare event (no proliferating cell nuclear Ag-positive F480+ cells were seen in the liver sections analyzed), we conclude that increased macrophage recruitment is unlikely to have accounted for the increased macrophage population seen in the apoE-deficient mice.

A summary of the macrophage population dynamics in the liver from WT and apoE-deficient mice is shown in Fig. 3D. ApoE deficiency has significantly perturbed the homeostasis of the macrophage population, resulting in a small but statistically significant increase in macrophage recruitment, a larger increase in the number of live macrophages but the most significant effect is the dramatic increase in the number of apoptotic bodies. Based on this analysis, we conclude that clearance of apoptotic bodies is decreased in the liver of apoE-deficient mice in vivo as well as in vitro.

Macrophage population dynamics in other tissues

Cryosections were prepared from the brain (left hemisphere) and left lung of the same mice from which liver samples had been taken. As in the liver, apoE deficiency increased the size of the alveolar macrophage population (Fig. 4A), with the dominant factor contributing to this increase being the accumulation of dead and dying cells (Fig. 4B). The magnitude of the effects seen in the lung were very similar to those seen in liver.



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  		FIGURE 4. Macrophage populations in mouse lung and brain. A, The number of F4/80+ cells (mostly alveolar macrophages) in 10 sections from the lung from six mice of each genotype expressed as a percentage of the total number of nuclei analyzed. B, The number of TUNEL+F4/80+ cells in the same section as in A expressed as a percentage of the number of F4/80+ cells. C, The number of MHC class II+ cells (predominantly microglia) in 10 sections from the brain of six mice of each genotype expressed as a percentage of the total number of nuclei analyzed. D, The number of TUNEL+MHC class II+ cells in the same sections as in C expressed as a percentage of the number of MHC class II+ cells. In each case, values are mean ± SEM for six animals.

 
It was not possible to use the same Ab markers to characterize the brain tissue macrophage population (the microglia) because they only stained weakly with the F4/80 mAb, consistent with previous findings (42). However, an Ab against MHC class II (which only stains a subset of the F4/80+ cells in liver and lung) stained the microglia as previously described (42). ApoE deficiency increased the number of MHC class II+ cells in the brain (Fig. 4C), albeit to a lesser extent than seen for the F4/80+ population in lung and liver. However, consistent with our observations in the other tissues, both the absolute number and the proportion of MHC class II+ cells which were TUNEL+ were markedly increased (Fig. 4D). Similar results were obtained using an Ab against CD14 (data not shown) to detect the microglial population, although CD14 may be selectively detecting newly recruited macrophages or activated microglia (43).

Taken together, these observations suggest that the attenuation of apoptotic body uptake in the absence of apoE which we observed in vitro has resulted in a systemic accumulation of uncleared apoptotic bodies at equilibrium in apoE-deficient mice and that there has been a small, but statistically significant, increase in macrophage recruitment to a range of tissues, possibly in response to the defect in clearance of apoptotic cells.

Effect of apoE deficiency on markers of inflammation in the liver

Quantitative immunofluorescence was used to measure the relative levels of two markers of inflammation in the liver from apoE-deficient mice and their WT littermates. The cytokine TNF-{alpha} is up-regulated during acute inflammation, but is normally present at only very low levels in healthy liver, predominantly in the perivascular regions (Fig. 5A). Staining for TNF-{alpha} was 1.5-fold higher in apoE-deficient mice compared with that of WT littermates (p < 0.05, Mann-Whitney U test; Fig. 5B). Although the levels of this cytokine were low in both groups, apoE deficiency led to a statistically, and possibly biologically, significant change in TNF-{alpha} levels.



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  		FIGURE 5. Markers of inflammation in mouse liver. A, A fluorescence micrograph of a typical section of liver from a WT mouse stained for TNF-{alpha}. Most regions of the section contain little or no detectable TNF-{alpha}, but cells in the region of the small blood vessels stain strongly positive. Scale bar, 25 µm. B, TNF-{alpha} staining in 10 sections of liver from each of six mice of each genotype measured by quantitative immunofluorescence procedures as previously described. C, A fluorescence micrograph of a typical section of liver from a WT mouse stained for fibrinogen. High levels of staining are seen in the central veins of each functional unit within the liver as well as in the sinusoids. Scale bar, 25 µm. D, Fibrinogen staining in 10 sections of liver from each of six mice of each genotype measured by quantitative immunofluorescence procedures as previously described. In each graph, values are mean ± SEM for six mice.

 
Staining for the hepatocyte product fibrinogen (Fig. 5C) was also increased by apoE deficiency (p < 0.05, Student’s unpaired t test; Fig. 5D). Although primarily involved in blood clotting, fibrinogen is known to be a positive acute phase reactant (i.e., a gene product whose levels are increased during acute inflammatory responses). We conclude that two unrelated markers of systemic inflammation (TNF-{alpha} and fibrinogen) are elevated in apoE-deficient mice compared with their controls, although our present studies cannot determine whether increased levels of TNF-{alpha} and fibrinogen precede or follow the systemic increase in tissue macrophage density.

Effect of plasma cholesterol concentration on macrophage population dynamics

In vivo, apoE deficiency results in a very significant misregulation of lipoprotein metabolism (2, 3). To determine whether alterations in lipoprotein metabolism might indirectly affect macrophage population dynamics, we used two strategies independent of apoE deletion to alter lipoprotein metabolism. First, we examined the impact of feeding the WT mice a high cholesterol diet for 10 wk, which resulted in an increase in both plasma LDL and VLDL cholesterol concentrations and a decrease in high-density lipoprotein cholesterol (Fig. 6A), so that the lipoprotein profile of the fat-fed C57BL/6 mice more closely resembled the profile of the apoE-deficient mice, as previously observed (37). At the end of this period, there was no difference in the liver macrophage population (p = 0.94, Student’s unpaired t test vs WT mice on a normal chow diet; Fig. 6B). Similarly, genetic deletion of the LDLR had only a marginal effect on the macrophage population in liver (p = 0.07, Student’s unpaired t test; Fig. 6D) and no effect on the populations in brain and lung (p = 0.88, Student’s t test; Fig. 6D), despite causing a defect in lipoprotein metabolism comparable in magnitude to that seen following deletion of apoE (Fig. 6C) as observed previously (32). We conclude that systemic alterations in lipoprotein metabolism are unlikely to cause the changes in macrophage population dynamics seen in apoE-deficient mice, and furthermore that apoE-LDLR interactions are unlikely to mediate the attenuation of apoptotic body clearance we observed.



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  		FIGURE 6. Effect of altered lipoprotein metabolism on macrophage populations. A, Lipoprotein profile of pooled sera from six WT mice sacrificed at 22 wk of age, fed either a high fat diet ({circ}) or normal chow diet ({blacksquare}) for 10 wk. The lipoprotein profile from pooled sera from six apoE-deficient mice is shown (plain line) for comparison. In this protocol, VLDL elutes before fraction 10, LDL between fractions 10 and 20, and HDL after fraction 20. B, The number of F4/80+ cells expressed as a percentage of the total number of nuclei analyzed in 10 sections of liver from each of six mice in each group. C, Lipoprotein profile of pooled sera from six LDLR-deficient mice on a normal chow diet throughout. The lipoprotein profile from pooled sera from six apoE-deficient mice is shown (plain line) for comparison. D, The number of F4/80+ cells expressed as a percentage of the total number of nuclei analyzed in 10 sections of liver ({cjs2108}) or lung ({cjs2098}) from each of six mice of each genotype. In each graph, values are mean ± SEM for six mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materils and Methods
 Results
 Discussion
References
 
We have demonstrated that complete deficiency of apoE protein in macrophages specifically attenuates the ingestion of apoptotic cells in vitro, without affecting general phagocytosis function. This defect results in a marked increase in the accumulation of apoptotic cells and fragments in a range of tissues in apoE-deficient mice in vivo and also in a larger population of live macrophages in these tissues. This, in turn, is associated with a systemic increase in proinflammatory markers, including TNF-{alpha} and fibrinogen. Although genetic deletion of apoE has previously been reported to promote macrophage recruitment to the blood vessel wall (3, 44, 45), this has generally been assumed to be an indirect response to local lipid deposition rather than directly a result of apoE deficiency. In this study, we show that alterations in lipid metabolism are unlikely to be responsible for the effects we have observed and provide the first direct evidence for a systemic effect of apoE on tissue macrophage recruitment which is independent of lipoprotein metabolism, resulting from impaired uptake of apoptotic cell remnants.

The molecular mechanism by which apoE deficiency attenuates the uptake and clearance of apoptotic bodies is unclear. Since deletion of the LDLR does not replicate the effects of apoE deletion on macrophage population dynamics, we can conclude that apoE-LDLR interactions are unlikely to be important. More likely, the effect of apoE is mediated through the LRPs, which have been implicated in other apoE signaling pathways (1, 7, 13, 14). Our observations do make clear, however, that the phenotype of the LDLR-deficient mice and the apoE-deficient mice, both of which have been used as animal models of atherosclerosis, are likely to be different particularly when considering the inflammatory aspect of vascular disease processes, which may explain the marked difference in the response of these two mouse lines to high doses of statins which was reported recently (46).

Rather than a direct role, for example, by tethering the apoptotic cell to the engulfing macrophage, it seems more likely that apoE indirectly affects macrophage uptake, for example, by modulating the gene expression of other factors critical for apoptotic clearance, particularly since exogenous soluble apoE is capable of overcoming the defect due to endogenous apoE deficiency. This is also consistent with previous reports that apoE is not detectable on the cell surface of macrophages (47). Various gene products are now known to be essential for apoptotic body clearance including the mer tyrosine kinase (31) and the ATP-binding cassette protein ABC-A1 (48), and it remains plausible but unproven that apoE modulates the clearance process by changing the expression of proteins such as these.

Recently, Koistinaho et al. (49) demonstrated that apoE-deficient astrocytes internalize amyloid {beta} peptide deposits much less well than do WT astrocytes. The characteristics of this apoE-dependent internalization process that they describe are similar to the characteristics of the apoE-modulated uptake of apoptotic bodies we describe here, and it is plausible that a common molecular mechanism underlies both processes. Both studies provide strong independent evidence for an unanticipated role of apoE as a modulator of diverse phagocytotic functions.

Another question raised by our observations is the nature of the association between reduced clearance of apoptotic bodies and the enlargement of the tissue macrophage population in a range of tissues. It is possible that these two effects of apoE deficiency are independent, but is seems more plausible to suggest that the increase in macrophage recruitment is a consequence of accumulating apoptotic bodies. Not only is the clearance of such cellular debris an important function of tissue phagocytes, making it an appealing hypothesis to suggest that phagocyte number should be linked to the amount of debris awaiting clearance, but more recently a molecular mechanism to explain such a link has been uncovered. Huynh and coworkers (50) demonstrated that macrophages secrete the cytokine TGF-{beta} upon taking up apoptotic bodies, at least in the lung and peritoneum, although a different mechanism seems to be operative in brain (51). Since TGF-{beta} is a major anti-inflammatory cytokine acting to suppress monocyte recruitment into a range of tissues (52), this provides a mechanistic link between apoptotic clearance and macrophage numbers: if uptake of apoptotic bodies is impaired, then TGF-{beta} secretion will be reduced and macrophage numbers will increase. Consistent with this proposed pathway, summarized in Fig. 7, we have previously observed a reduction in the level of TGF-{beta} activity in various tissues from apoE-deficient mice (53).



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  		FIGURE 7. A model of macrophage population dynamics in mouse liver. Processes increasing the equilibrium population of mature macrophages (deep pink cells; F4/80+) include recruitment of monocytic precursors across the vascular endothelium (blue cells), differentiation via M1/70+F4/80+ newly recruited macrophages (light pink cells), and proliferation (data not shown). Processes reducing the equilibrium population of mature macrophages include emigration across the endothelium into lymph or blood and death, either by apoptosis or necrosis (TUNEL+F4/80+ cells; stippled pink cell) followed by clearance via phagocytosis. We demonstrated that apoE deficiency attenuates clearance of apoptotic cell remnants (red box). Huynh et al. (50 ) have demonstrated that release of TGF-{beta} is stimulated in macrophages taking up apoptotic cell remnants. This TGF-{beta} would be expected to inhibit recruitment of monocytes across the vascular endothelium (black line), resulting in a coupling of the size of the tissue phagocyte population with the number of cell remnants awaiting clearance.

 
Irrespective of the mechanism resulting in increased monocyte recruitment and increased macrophage density, apoE deficiency also results in a mild proinflammatory phenotype. The levels of TNF-{alpha} and fibrinogen are somewhat increased by apoE deficiency. Whether these changes in the levels of central proinflammatory signaling molecules such as TNF-{alpha} are responsible for the altered macrophage population dynamics might be open to investigation: mice deficient in TNF-{alpha} have already been generated (54), and if TNF-{alpha} levels are, in part, responsible for transducing the apoE signal into changes in macrophage numbers, then we will also observe similar alterations in macrophage population dynamics in TNF-{alpha}-deficient animals to those reported here. Alternatively, if tissue macrophage dynamics are unaffected by TNF-{alpha} deletion, then we can conclude that a systemic proinflammatory change in the levels of molecules such as TNF-{alpha} and fibrinogen is likely to be a consequence, rather than a cause, of the increase in tissue macrophage numbers.

Although a number of questions remain unanswered, our studies provide direct evidence of a central physiological mechanism that provides a plausible explanation for the observed association between apoE genotype and a range of diseases with a macrophage-rich inflammatory component. Our working hypothesis is that apoE is required for efficient clearance of apoptotic bodies and that different apoE genotypes are associated with slight differences in the clearance rates for apoptotic bodies. Unfortunately, subtle differences between apoE isoforms would not be detectable by our current in vitro assays which are only powered to detect a 30% or so difference such as that caused by complete apoE deficiency. However, over time in vivo, even a small decrease in apoptotic body clearance would result in increased flux through the monocyte/macrophage pathway and contribute to a systemic proinflammatory shift in phenotype. One sequela of such a shift will be a fibrotic tendency that contributes to the loss of functional tissue architecture that is a hallmark of Alzheimer’s disease and atherosclerosis. The histological similarity between the F4/80+TUNEL+ cells in the liver, brain, and lung of apoE knockout mice with extracellular Lewy bodies in human brain (which are thought to result from persistent apoptotic cells) lends credence to this hypothesis.

Once the molecular pathways by which apoE exerts these functions independent of lipoprotein metabolism become clear, it may be possible to devise therapies that address this mechanism. Studies by Laskowitz et al. (18) already provide a lead in this direction: they have shown that a receptor-binding peptide derived from apoE can inhibit microglial activation in response to acute proinflammatory stimuli. Another approach would be to stimulate apoE production by macrophages, and activators of the transcription factors PPAR-{gamma} and LXR-{alpha} are both known to stimulate apoE production (55, 56) and to protect against early atherosclerosis in mice (56, 57). Pharmacological regulation of the apoE signaling pathway may one day offer the ability to regulate tightly the resting tissue macrophage population (independent of, or in addition to, regulation of lipoprotein metabolism) and so impact the incidence of a wide range of prevalent diseases with a genetic association at the apoE locus.


   Acknowledgments
 
We are grateful to Dr. David Mosedale for his assistance with the quantitative immunofluorescence methodology.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was funded by grants from the British Heart Foundation, Wellcome Trust, and NeoRx Corp (Seattle, WA) to D.J.G., who is a British Heart Foundation Senior Research Fellow. Back

2 Address correspondence and reprint requests to Dr. David J. Grainger. Department of Medicine, Addenbrooke’s Hospital, Box 157, University of Cambridge, Hills Road, Cambridge, CB2 2QQ, U.K. E-mail address: djg15{at}cam.ac.uk Back

3 Abbreviations used in this paper: apoE, apolipoprotein E; LDL, low-density lipoprotein; LDLR, LDL receptor; LRP, LDL receptor-related protein; VLDL, very LDL; WT, wild type. Back

Received for publication May 12, 2004. Accepted for publication August 24, 2004.


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