Macrophage Phenotype in Mice Deficient in Both Macrophage-Colony–Stimulating Factor (Op) and Apolipoprotein E

From the Sir William Dunn School of Pathology, University of Oxford, UK (W.J.S. de V., E.D., S.G.); and the Laboratory of Biochemical Genetics and Metabolism, The Rockefeller University, New York, NY (J.D.S., M.M., H.M.D.). Dr de Villiers is now with the Division of Gastroenterology, Department of Medicine, University of Kentucky Medical Center, Lexington, KY.

Correspondence to Dr Willem J.S. de Villiers, Division of Gastroenterology, Department of Medicine, University of Kentucky Medical Center, Lexington, KY 40536-0084. E-mail wdevil0{at}pop.uky.edu

	Abstract

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Abstract—Mice deficient in both macrophage-colony–stimulating factor (M-CSF, op) and apolipoprotein E (apoE) have elevated cholesterol levels but are protected from atherosclerosis. To assess the contribution of macrophage (M) phenotypic heterogeneity and scavenger receptor (SR-A) expression to this seeming paradox, we characterized the M phenotype by immunohistochemistry in these animals. Lesion size was determined in animals fed a chow or Western-type diet, and lipoprotein clearance studies were performed in vivo. Op0/E0 mice have fourfold smaller aortic root lesions than op2/E0 animals despite 2.5-fold higher total plasma cholesterol levels. Ms in atherosclerotic lesions of op2/E0 mice constitute a predominantly recruited and M-CSF–dependent population. In addition, Ms in different locations in plaques show phenotypic heterogeneity. SR-A expression in op0/E0 mice is reduced in proportion to the decrease in M numbers, and M-CSF is thus not an essential requirement for SR-A expression in vivo. M-CSF–deficient mice degrade injected AcLDL , showing an adequate level of SR-A activity present in vivo. In contrast, ß-VLDL clearance in op0/E0 mice is decreased, implicating monocytes/Ms in its catabolism. There is prominent lipid accumulation in op2/E0 Kupffer cells and hepatocytes but not in M-CSF–independent Kupffer Ms from op0/E0 mice. SR-A, while abundantly expressed on both Kupffer cells and sinusoidal endothelial cells in op2/E0 mice, remains mainly on sinusoidal endothelial cells in op0/E0 mice. This may explain preservation of SR-A activity in these animals. Our findings clearly illustrate the importance of both M-CSF and M-CSF–dependent monocytes/Ms in maintaining cholesterol homeostasis and in atherogenesis.

Key Words: macrophages • macrophage-colony-stimulating factor • apolipoprotein E • atherosclerosis

	Introduction

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The arterial fatty streak is an early and reversible precursor to advanced fibroproliferative atherosclerotic lesions.¹ These early lesions contain mostly monocyte-derived Ms that have taken up modified lipoproteins to become lipid-enriched foam cells. In addition to M foam cells, human lesions also contain T cells and smooth muscle–derived foam cells,¹ and an essential role of monocyte-derived Ms in atherosclerosis remains unproven.

ApoE-deficient mice are an attractive model system in which to evaluate this question. These mice are hypercholesterolemic and develop atherosclerosis spontaneously on a low-fat chow diet and in an accelerated fashion on a high-fat Western-type diet.² Atherosclerotic lesions in apoE-deficient mice occur throughout the aortic tree, progress with age from early fatty streaks to complex fibrous lesions with necrotic cores, and are found at the same sites of predilection as human lesions.^{3 4}

M-CSF and its effects on M development and function play a key role in atherogenesis. M-CSF injections reduce cholesterol levels in rabbits, primates, and humans^{5 6 7} and decrease atherosclerosis and carrageenan-induced granuloma lipid accumulation in Watanabe hyperlipidemic rabbits.^{7 8} The M-specific membrane molecule macrophage scavenger receptor (or SR-A) has been proposed to have an important in vivo role in atherogenesis through its involvement in foam cell formation.⁹ Recent evidence supports a proatherogenic role for SR-A in that SR-A–deficient mice crossed onto an apoE-deficient background were relatively protected from atherosclerosis.¹⁰ M-CSF potently and selectively increases SR-A expression, stability, and endocytic and adhesion functions in murine Ms in vitro.¹¹

The osteopetrotic (op/op) mouse has a spontaneously derived recessive mutation in the gene encoding M-CSF and the phenotype has been well characterized.^{12 13 14} The op defect in the M-CSF gene is a frameshift mutation leading to the complete absence of M-CSF activity in the serum and tissues.¹⁵ Osteopetrotic mice lack osteoclasts, resulting in impaired bone remodeling and skeletal deformities, the most severe being a deficiency in tooth eruption, so that young op/op mice require a soft diet to survive.¹⁶ Hematologically, op/op mice have normal hematocrits and granulocyte counts but markedly decreased blood monocytes and peritoneal Ms.¹⁷ The op/op mouse thus provides an opportunity to examine the contribution of M-CSF to specific M populations in vivo, with particular regard to levels of SR-A expression.

Immunohistochemical studies in op/op mice have shown that other M populations apart from osteoclasts are critically dependent on M-CSF in vivo.¹³ These M-CSF–dependent populations include peritoneal Ms, splenic marginal zone metallophils, and lymph node subcapsular sinus Ms. Liver Ms (Kupffer cells) are reduced but still readily identifiable; other M populations, including those within the thymic cortex, splenic red pulp, lymph node medulla, intestinal lamina propria, lung (alveolar Ms), and brain (microglia), as well as dendritic cells, remain present in substantial numbers and appear for the most part M-CSF independent.

Op/op mice were recently crossed onto the apoE-deficient background to determine the role of monocyte-derived Ms (and M-CSF) in atherogenesis in vivo.¹⁸ The double-mutant mice had an almost threefold increase in plasma cholesterol compared with apoE-deficient controls. Despite this severe hypercholesterolemia, proximal aorta atherosclerosis in these mice was significantly decreased. These findings were confirmed in a recent study describing a genetically more homogeneous population.¹⁹ Using a rabbit anti-mouse M polyclonal antiserum for immunohistochemistry, prominent and homogeneous M staining was revealed in small raised foam cell lesions from both op2/E0 and op0/E0 mice.¹⁸ This pan-M staining would not reveal any M functional heterogeneity in lesions. Interestingly, the reduced monocyte number in the op0/E0 mice was shown statistically not to affect lesion formation independently,¹⁸ and a crucial factor may rather be the antiatherogenic functional modulation of those remaining M-CSF–independent Ms. Diminished SR-A expression, for example, may protect against foam cell formation and atherosclerosis in the doubly mutant mice.

To extend this work further, we characterized the development of atherosclerosis in apoE-deficient mice (E0; E for apoE followed by the number of wild-type alleles to describe the mouse genotype), as well as mice doubly mutant for the apoE gene and M-CSF genes (heterozygous and homozygous) (op1/E0; op0/E0: op2, op1, or op0; op for osteopetrotic followed by the number of wild-type alleles). Lesion size was measured in animals fed a chow diet for 1 year and in animals fed a Western-type diet for 12 weeks after weaning. We also examined tissues from 16-week-old mice fed a chow diet. M subpopulations in the op2/E0, op1/E0, and op0/E0 double-mutant crosses were characterized by immunohistochemistry of M membrane molecules with special emphasis on SR-A expression. In addition, lipoprotein clearance studies were performed in op2/E0 and op0/E0 mice in vivo with AcLDL and ß-VLDL to determine their functional phenotype.

	Methods

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Mice and Diet
The creation of the apoE-deficient mice used in this study has been described.² The C57BL/6x129 apoE-deficient female mice were bred to op heterozygous (op1) male mice (The Jackson Laboratory) on the C57BL/6xC3H background. Heterozygous E1 progeny were screened for the op mutation by a polymerase chain reaction assay as described,¹⁸ and the op1/E0 mice among them were interbred to generate op1/E0 animals that served as the parental genotype for all animals in the study. Offspring of the parental op1/E0 were op0/E0, op1/E0, and op2/E0 littermates and served as the subjects in the study. The mice were weaned at 4 weeks and maintained either on a chow diet (4.5% fat and 0.02% cholesterol by weight) or a Teklad adjusted Western-type diet (42% fat, 0.15% cholesterol, and 19.5% casein without sodium cholate by weight). Diet and water were provided ad libitum.

Immunohistochemistry
The animals were killed at 16 weeks and saline perfused. Organs (heart, thoracic and abdominal aorta, lungs, liver, spleen, gut, kidneys, and skin) were removed from op0/E0 (n=4), op1/E0 (n=4), and op2/E0 (n=4) littermates. Tissues were washed in PBS, placed in Tissue-Tek OCT compound (BDH-Merck), and snap-frozen in isopentane cooled by dry ice. Frozen sections were cut on a Leica cryostat (5 µm thick), collected onto 1.5% gelatinized slides, air dried for 1 hour, and stored at -20°C for later use. Sections were fixed for 10 minutes in 2% paraformaldehyde in HEPES-buffered isotonic saline before staining.

The mAbs used in this study are listed in Table 1, along with their specificity, isotype, and appropriate references.^{20 21 22 23 24 25 26 27 28 29} Fixed sections were washed in PBS containing 0.1% vol/vol Triton X-100 and treated with 2% normal rabbit serum for 30 minutes. Sections were incubated for 90 minutes with primary antibody (10 µg/mL purified, or neat tissue hybridoma supernatant), PBS, or isotype-matched control mAb. Endogenous peroxidase activity was blocked by incubation of sections with 0.01 mol/L glucose, 0.001 mol/L sodium azide, 40 U glucose oxidase in 100 mL phosphate buffer for 15 minutes at 37°C.³⁰ Affinity-purified, mouse-adsorbed, biotin-ylated second Ab (Vector Labs) was used at 1% for 45 minutes followed by avidin-biotin-peroxidase complex (ABC elite, Vector) according to the supplier's recommendation.³¹ The presence of antigen was revealed by incubation with 0.5 mg/mL diaminobenzidine (Polysciences, Inc) and 0.024% H₂O₂ in 10 mmol/L PBS imidazole, pH 7.4. Counterstaining was with cresyl fast violet acetate, and stained cells were dehydrated and mounted in DPX (BDH-Merck). Representative photographs were taken using a blue filter (Kodak, Wratten, gelatin filter No. 47) which intensifies the brown precipitate. Serial sections adjacent to sections in which immunostained morphology was recorded were stained with oil red O for the detection of lipids.³²

In Vivo Lipoprotein Turnover Study
AcLDL was prepared from human LDL as previously described.³³ ß-VLDL was floated by ultracentrifugation of pooled apoE-deficient mouse plasma overlaid with PBS. Lipoprotein (100 µL) was labeled by overnight incubation at 37°C with 10 µL of [³H]cholesteryl oleyl ether (Amersham), dried under nitrogen, and 1 µL of recombinant cholesterol ester transfer protein (generously provided by Alan Tall, Columbia University). Labeled lipoproteins were separated from unincorporated label by gel filtration. Lipoproteins were injected into the femoral vein of sodium pentobarbital–anesthetized mice, and blood was removed at the indicated times from the retroorbital plexus. For each mouse, the remaining plasma radioactivity was normalized to the radioactivity in the initial bleeding. This method of normalization was used, instead of normalization to the calculated injected radioactivity, because preliminary experiments revealed that this method was more reproducible due to difficulty in quantitatively injecting the labeled sample without any loss.

Atherosclerosis, Cholesterol, and Monocyte Differential Assays
These assays were performed as previously described.¹⁸

	Results

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Development of Atherosclerosis in Op2/E0 and Op0/E0 Mice
We confirmed and extended the initial finding of decreased atherosclerosis in chow-fed op0/E0 mice analyzed at 16 weeks.¹⁸ We further analyzed atherosclerosis in 1-year-old chow-fed op0/E0 and op2/E0 littermates using an en face method along the entire aorta.³⁴ Two males and one female of each genotype were analyzed. Oil red O–staining lesions covered 31.8%±10.2% of the aortic surface in the op2/E0 mice, while in the op0/E0 mice, the lesions covered only 3.2%±2.7% of the surface (P<.01 by a two-tailed t test).

In a separate study, op0/E0 and op2/E0 littermates were weaned at 3 weeks of age onto a Western-type diet (42% fat and 0.15% cholesterol by weight). At 12 weeks of age, the mice were killed and the lesion areas in the aortic root were determined as previously described.¹⁸ The op2/E0 mice had very large foam cell lesions, of which many were beginning to form fibrous caps. The mean lesion area was about 250 000 µm² per section. The aortic root lesions in the op0/E0 mice were about fourfold smaller than in the op2/E0 mice, irrespective of gender (Fig 1). This phenomenon occurred despite increased total plasma cholesterol in the op0/E0 mice. Total plasma cholesterols were 2152±424 mg/dL in the op0/E0 (n=18) and 1420±39 mg/dL in the op2/E0 mice (n=25, P<.0001). Smaller lesions in the op0/E0 mice were associated with a decreased monocyte differential, 3.30%+1.79%, (n=18) compared with 10.32%+2.49% in the op2/E0 mice (n=25, P<.001).

View larger version (26K): [in this window] [in a new window]   Figure 1. Atherosclerotic lesion areas in op0/E0 and op2/E0 mice. Mice were fed a high-fat Western-type diet and killed at 12 weeks of age. Mean lesion area in the aortic root was determined in 5 op0/E0 males and 15 op2/E0 males, *P<.01 compared with op2/E0 males by a two-tailed t test. Mean lesion area was also determined in 13 op0/E0 females and 10 op2/E0 females, **P<.001 compared with op2/E0 females by a two-tailed t test.

Characterization of M Phenotypes in Atherosclerotic Lesions in Op2/E0, Op1/E0, and Op0/E0 Mice
Sixteen-week-old mice fed a chow diet were killed and examined. Every op1/E0 and op2/E0 mouse analyzed had atheromatous lesions in the aortic root, situated on the valve cusps and in the areas between valve cusps. These lesions comprised a spectrum of both simple raised foam cell type and larger, more developed fibroproliferative plaques. In Fig 2, immunohistochemistry from representative advanced lesions (as shown by oil red O staining) in an op2/E0 mouse is shown. Recruited Ms within the lesions prominently expressed the M-specific late endosomal membrane glycoprotein macrosialin.³⁵ This mostly intracellular membrane molecule (homologous to human CD68³⁶) was present throughout the lesion, including its base, shoulders, and necrotic core.

View larger version (87K): [in this window] [in a new window]   Figure 2. M phenotype analysis in atherosclerotic lesions in op2/E0 and op1/E0 mice. A representative advanced lesion from an op2/E0 animal fed an atherogenic diet for 16 weeks was stained with oil red O, FA-11, 2F8, 5C6, 2.4G2, and TIB120. Isotype-matched negative controls not shown. Magnification x100. A, Oil red O; B, macrosialin; C, SR-A; D, CR3; E, FcRII; and F, MHC class II. M phenotypic heterogeneity is present and tabulated in Table 2.

CD11b (CR3) and SR-A, examples of M membrane molecules that have both endocytic and adhesive functions, were also abundantly expressed in the lesion. CD11b is generally considered a marker of recently recruited Ms,³⁷ whereas SR-A expression accompanies monocyte to M differentiation.³⁰ Different functional subsets of lesional Ms are thus characterized by these markers. Interestingly, in view of its envisaged role in foam cell formation, marked SR-A staining was present on foam cell membranes in smaller immature lesions in op1/E0 animals (data not shown). CD44, another adhesion molecule possibly involved in M recruitment and retention within the atheromatous microenvironment, was homogeneously expressed in the lesion and immediate surrounding areas (not shown). Staining with an irrelevant isotype control antibody, CAMPATH-1G, was negative.

Ms in the atherosclerotic lesions also expressed FcRII (CD32). Oxidized lipoprotein epitopes are present in the lesions of E0 mice, and sera from these mice contain high titers of autoantibodies to oxidized lipoproteins.³⁴ This molecule may therefore be functionally relevant in IgG-mediated phagocytosis by lesional Ms. As dendritic cells (NLDC145) and T lymphocytes (CD4 and CD8) (not shown) were virtually absent, prominent MHC class II staining indicates the presence mainly of activated lesional Ms. The reported predominant Th1 phenotype of T lymphocytes in the atherosclerotic plaque may implicate IFN-–dependent upregulation of MHC class II expression on these Ms.³⁸

In contrast to these markers of a recruited M population, F4/80 and sialoadhesin expression levels in the lesions were unimpressive and consistent with their status as resident and stromal M markers, respectively.³⁷ Lesions in op1/E0 animals showed a similar M presence and phenotype as op2/E0 mice; and a gene dosage effect of M-CSF deficiency could not be demonstrated (data not shown). The distribution of M membrane molecules within op2/E0 atherosclerotic lesions is tabulated in Table 2.

View this table: [in this window] [in a new window]   Table 2. Distribution of M Membrane Molecules in op2/E0 Atheromatous Lesions

All the lesions in op0/E0 animals were at the aortic root on the valve cusps. The modulation in E0 atherogenesis brought about by the op/op mutation was readily apparent (Fig 3). Oil red O staining revealed that M-CSF deficiency resulted in fewer and smaller lesions in op0/E0 mice; these were of mostly early foam cell type and had not progressed to more advanced stages and were primarily located on valve cusps. When compared with op2/E0 mice, the decrease in atherosclerotic lesions was paralleled by an overall reduction in Ms present in lesions. It was not possible to ascertain whether SR-A expression was disproportionately reduced in op0/E0 lesional Ms in comparison to expression levels of macrosialin, CD11b (CR3), FcRII, MHC class II, or CD44.

View larger version (96K): [in this window] [in a new window]   Figure 3. M phenotype analysis in atherosclerotic lesions in op0/E0 mice. A representative early foam cell–type lesion from an op0/E0 animal is shown, stained with oil red O, FA-11, 2F8, 5C6, TIB120, and 2.4G2. Isotype-matched negative controls not shown. Magnification x100. Lesions were fewer and less advanced than op2/E0 animals and located exclusively on valves and valve cusps. A, Oil red O; B, macrosialin; C, SR-A; D, CR3; E, MHC class II; and F, FcRII.

The Ms characterizing the aortic root lesions in op2/E0 mice are therefore predominantly recruited and M-CSF dependent. Some Ms still remain as an integral component of the smaller lesions present in op0/E0 mice, and these represent an M-CSF–independent population. While the M-CSF deficiency in op0/E0 animals affected M functional heterogeneity within atherosclerotic lesions, this is unlikely due to a selective event and may rather reflect heterogeneity among M populations in the whole animal in general.

Myocardial Interstitial Ms in Op2/E0 and Op0/E0 Mice
Numerous Ms were present in the op2/E0 myocardial interstitium, as was evident from immunostaining of the M-specific molecules macrosialin and SR-A, as well as CD44, MHC class II, CR3, and FcRII molecules (data not shown). These Ms have the characteristics of a recruited and not resident population, as suggested by the low expression of F4/80 and sialoadhesin. In contrast, expression of M membrane molecules, including SR-A, was markedly reduced in op0/E0 animals (data not shown). This would suggest a decrease in the interstitial myocardial M population as a whole in op0/E0 mice, indicating its M-CSF–dependent nature.

In Vivo Turnover of Lipoproteins in Op2/E0 and Op0/E0 Mice
Sixteen-week-old op0/E0 mice fed a chow diet were previously shown to have smaller atherosclerotic lesions than their op1/E0 or op2/E0 littermates, despite having 2.5-fold increased levels of plasma cholesterol.¹⁸ The increased cholesterol is primarily due to an increased level of cholesteryl ester–enriched ß-VLDL.^{18 39} Since M-CSF has been reported to induce SR-A mRNA,¹¹ the hypothesis that the increased plasma cholesterol in the op0/E0 mice is associated with decreased SR-A activity was tested. Op0/E0 and op2/E0 mice were injected IV with [³H]AcLDL, and plasma radioactivity was determined from retroorbital bleedings and normalized to the initial bleeding immediately after injection. Both types of mice degraded this type of scavenger receptor ligand extremely rapidly, with single-phase kinetics and a half-life of about 1.5 minutes (Fig 4A). To determine if the increased plasma ß-VLDL was associated with decreased clearance of this material, [³H]ß-VLDL was prepared from E0 mice and injected into both types of mice. The half-life of [³H]ß-VLDL was about 2 hours in the op2/E0 mice and about 5 hours in the op0/E0 mice (Fig 4B). However, the plasma disappearance of this material appeared to have a faster and a slower phase, with only the slower phase, apparent after 1 hour, showing a difference between the two groups of mice.

View larger version (15K): [in this window] [in a new window]   Figure 4. In vivo turnover of lipoproteins in op0/E0 and op2/E0 mice. A, AcLDL turnover. 3 op2/E0 and 5 op0/E0 mice were injected with [3H]AcLDL and bled immediately, 30, 60, and 120 seconds after injection. Each point represents the mean of the fraction of radioactivity remaining in the plasma compared with the initial point. B, ß-VLDL turnover. Op2/E0 and op0/E0 mice (4 each) were injected with [3H]ß-VLDL and bled after 2, 5, 15, 30, 60, 120, 180, 240, and 300 minutes. Each point represents the mean of the fraction of radioactivity remaining in the plasma compared with the initial 2-minute time point.

M Populations in Noncardiovascular Organs of Op2/E0 and Op0/E0 Mice
The liver in op2/E0 mice appeared macroscopically enlarged and fatty and oil red O staining confirmed marked foamy lipid accumulation in both Kupffer cells and hepatocytes (Fig 5). Immunohistochemical analysis of hepatic Ms from op2/E0 and op0/E0 mice is shown in Fig 6. The Kupffer cell population expressed macrosialin, SR-A, and CD44 (not shown) prominently. SR-A staining of sinusoidal endothelium was more apparent than previously described,³⁰ most likely due to differences in tissue preparation and immunostaining techniques used in this study. The prominent expression of SR-A by sinusoidal ECs (and its possible functional relevance in clearing atherogenic lipoproteins) contrasts markedly with its complete absence from ECs elsewhere, including myocardial endothelium. The phenotypic status of Kupffer cells as a mature resident M population was confirmed by the lack of CD11b (CR3) expression (not shown) and low levels of sialoadhesin staining.

View larger version (98K): [in this window] [in a new window]   Figure 5. Lipid accumulation in livers of op2/E0 and op0/E0 mice. Liver sections from op2/E0 and op0/E0 animals were stained with oil red O. Magnification x100. In op2/E0 animals, both Kupffer cells and hepatocytes accumulated lipid, while only Kupffer cells (reduced in number) in op0/E0 mice stained oil red O positive. A, Op2/E0 and B, op0/E0.

View larger version (183K): [in this window] [in a new window]   Figure 6. Immunohistochemistry of livers from op2/E0 and op0/E0 mice. Liver tissues were obtained from op2/E0 and op0/E0 animals and stained with FA-11 and 2F8. Magnification x100. FA-11 stained Kupffer cells, and both the reduction in absolute numbers and foamy nature of op0/E0 Kupffer cells are clearly apparent. SR-A was present on Kupffer cells and sinusoidal endothelium in op2/E0 mice; in op0/E0 mice, expression was maintained by sinusoidal ECs, but not Kupffer cells. A and B, FA-11; C and D, 2F8.

In contrast, the livers from op0/E0 mice appeared nonfatty despite the extremely high levels of plasma cholesterol. Hepatocytes showed little evidence of lipid accumulation, but Kupffer cells (reduced in number) were still oil red O positive and remained capable of lipid uptake (Fig 5). The M-CSF–dependent reduction in Kupffer cells was further demonstrated by the decrease in macrosialin and SR-A staining (Fig 6), and foamy Ms were sparsely grouped throughout the hepatic architecture. SR-A expression remained well preserved on the sinusoidal EC population, which did not appear foamy. As these cells lack the M-CSF receptor (c-fms), it is not surprising that M-CSF deficiency did not affect SR-A expression on sinusoidal ECs in vivo.⁴⁰ Hepatic SR-A expression in op0/E0 animals was thus reduced, but not disproportionately so in comparison with other Kupffer cell markers.

In the small intestine lamina propria, Ms from op2/E0 or op0/E0 did not accumulate lipid (by oil red O staining) or appear foamy (Fig 7). The M-CSF–independent nature of this differentiated M population was shown by the abundant expression of the antigens macrosialin, CD11b (Fig 7), FcRII, F4/80, and sialoadhesin (not shown) in both op2/E0 and op0/E0 animals; and the unchanged M population size in op0/E0 mice. Interestingly, SR-A expression in op0/E0 mice appeared selectively decreased in this M population compared with op2/E0 controls.

View larger version (116K): [in this window] [in a new window]   Figure 7. Characterization of intestinal lamina propria Ms in op2/E0 and op0/E0 mice. Small intestinal tissues were stained with FA-11, 5C6, and 2F8. Magnification x100. This M population seemed for the most part M-CSF independent. SR-A staining, however, appeared reduced in op0/E0 lamina propria Ms compared with op2/E0 animals. A and B, macrosialin; C and D, SR-A; and E and F, CR3.

Analysis of the spleens revealed the combined absence of CD11b, sialoadhesin, and SR-A staining in marginal zones of op0/E0 mice (not shown). This finding confirms the op0 status of the doubly mutant animals as splenic marginal zone metallophil Ms are absent in op0 mice.¹³ Absence of SR-A expression in this M-CSF–dependent marginal zone metallophil population indicated loss of a specific M subpopulation rather than a selective decrease in antigen.

	Discussion

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An unexpected finding from the previous study of Smith and colleagues¹⁸ was the significant inverse correlation between plasma cholesterol levels and aortic root lesion area in chow diet–fed op2/E0, op1/E0, and op0/E0 populations; implicating M-CSF gene dosage as the underlying cause for both phenomena. We confirmed and extended this finding in the current study. Twelve-week-old Western diet–fed op0/E0 mice had 4-fold smaller aortic root lesions than their op2/E0 littermates, and similarly, 1- year-old chow-fed op0/E0 mice had 10-fold less aortic lesion surface area than their op2/E0 controls as determined by an en face assay. These decreases in the extent of atherosclerotic lesion develop- ment occur despite the markedly increased total plasma cholesterol levels in the op0/E0 mice. Recently, in both a dietary and apoE-knockout model, M-CSF deficiency similarly resulted in significantly reduced atherosclerosis.¹⁹ M-CSF therefore affects susceptibility to atherosclerosis profoundly by either altering the number or functional status of monocyte-derived Ms.

The immunohistochemical findings depict a predominantly recruited M-CSF–dependent M population in atherosclerotic lesions in op2/E0 mice. The pan-M molecule macrosialin proved an excellent marker for the presence of intralesional Ms. Its ubiquitous presence and localization as a late endosomal membrane protein may suggest a role in M lipid loading; and recent work is consistent with its involvement in oxidized lipoprotein uptake.^{41 42} The prominent lesional expression of FcRII on Ms is also interesting as, in addition to IgG phagocytosis, a scavenging role, including the uptake of oxidized lipoprotein, has been proposed for this molecule.⁴³ CR3-positive cells within the lesions were all Ms, and not neutrophils, indicating monocyte-specific recruitment and rapid turnover within a chronic inflammatory focus.

The op0/E0 lesions were smaller and contained fewer M-CSF–independent Ms. These cells, similar to the Ms in op2/E0 lesions, represent a recruited population, as shown by an overall reduction in F4/80 and sialoadhesin levels. SR-A expression in op0/E0 mice is present but reduced in proportion to the decrease in M numbers. This observation would suggest that M-CSF is not an essential requirement for SR-A expression in vivo.

Lesional Ms in different locations in plaques showed phenotypic heterogeneity by immunohistochemical staining. SR-A was present throughout the lesion except for the necrotic core, while CR3 expression in the murine advanced lesion (op2/E0) was not only confined to the deeper layers but also occurred in the superficial layers and necrotic core. Almost all the Ms within lesions in op2/E0 and op0/E0 mice expressed macrosialin and MHC class II antigens. This finding agrees with the reported pattern of CD68 and the HLA-DR expression within human atherosclerotic lesions from the aorta and coronary and carotid arteries.⁴⁴ Ms within atherosclerotic lesions may also be heterogeneous with regard to expression of SR-A isoform (type I or II). The 2F8 mAb recognizes both types of murine SR-A, and this issue was thus not definitively addressed. Interestingly, type II SR-A seems the predominant murine form expressed in vivo,^{23 30} whereas no differential expression of human SR-A isoforms could be detected by immunostaining in M from various organs and atherosclerotic foam cells.⁴⁵

Lesional M foam cell functional heterogeneity may be influenced by (1) the duration of residence within the lesion; (2) the local microenvironment of stimulatory and inhibitory growth factor and cytokine signals; and (3) regional location within the lesion. For example, Ms at the shoulder of lesions express stromelysin activity selectively,⁴⁶ while within the necrotic core of advanced plaques M cell death may be either programmed (apoptosis)⁴⁷ or result from toxic accumulation of modified lipoprotein derivatives and nitric oxide or local depletion of M-CSF.⁴⁸ There is, however, little evidence that gene expression by lesional Ms in vivo changes cellular behavior or influences the initiation or progression of atherosclerosis.

Myocardial interstitial Ms also represented a recruited and M-CSF–dependent cell population. This is interesting because the myocardial interstitial M population in wild-type C57BL/6 mice fed a normal chow diet appeared less prominent in number and mostly resident in nature.⁴⁹ The recruitment of interstitial Ms into the myocardium of atherosclerosis-prone op2/E0 mice may be secondary to myocardial muscle injury, although no areas of focal necrosis or fibrosis due to myocardial infarction could be detected.

An intriguing finding in the current study was the prominent lipid accumulation in the livers of op2/E0 but not op0/E0 mice, despite a fourfold increase in plasma cholesterol in op0/E0 animals. The increased lipid uptake in op2/E0 Kupffer cells and hepatocytes contrasted markedly with a low level of uptake by Kupffer Ms in op0/E0 animals. The op0/E0 Kupffer cells represent a remnant M-CSF–independent M population that exhibits both fewer Ms and a nonselective decrease of M markers, including SR-A.

The increased plasma cholesterol in the op0/E0 mice was previously shown to be due primarily to increased levels of cholesterol-enriched ß-VLDL.¹⁸ This could be due to either an increased production rate or a decreased fractional catabolic rate. In the present study, we performed in vivo turnover studies to address this issue and to determine whether this effect might be mediated to SR-A. Both op0/E0 and op2/E0 mice degraded AcLDL equivalently and rapidly with a half-life of 1.5 minutes. However, after these studies were completed, Suzuki et al¹⁰ demonstrated that even in mice that have been made deficient in SR-A, the turnover in AcLDL is not impaired in in vivo turnover experiments. Thus, other molecules functionally related to SR-A, such as MARCO,⁵⁰ macrosialin,^{41 51} CD36,⁵² and SR-B1⁵³ may provide sufficient uptake pathways for AcLDL and similar ligands. Due to the redundancy in AcLDL receptors, it is not possible to demonstrate SR-A defects in vivo using AcLDL as a ligand. Therefore, despite our observations that the hepatic distribution of SR-A was different in op2/E0 and op0/E0 mice, with the former expressing SR-A on both Kupffer and sinusoidal cells and the latter expressing predominantly on sinusoidal ECs, no change in AcLDL turnover was detected. Although M-CSF upregulates SR-A expression on Ms in vitro, this study demonstrates that M-CSF is not essential for SR-A expression on Ms and sinusoidal endothelium in vivo.

In contrast to the AcLDL turnover, the ß-VLDL turnover studies revealed a difference in the two types of mice. Op2/E0 mice cleared the tracer with a half-life of 2 hours, as opposed to a 5-hour half-life in the M-CSF–deficient op0/E0 animals. The turnover of ß-VLDL in both types of mice appeared to have two kinetic phases, as is commonly observed in many lipoprotein turnover studies, with the more rapid phase accounting for 20% of the tracer disappearance in the first 30 minutes. This phase was similar in both types of mice and might be due to equilibration of the tracer in the extravascular compartment. The turnover of the ß-VLDL tracer then begins to diverge after this rapid phase, yielding a 2.5-fold increased ß-VLDL fractional catabolic rate for the op2/E0 mice over the M-CSF–deficient op0/E0 mice. In a steady state situation, the ß-VLDL production rate must equal the absolute ß-VLDL degradation rate, and while the production rate is independent of the ß-VLDL pool size (zero order reaction), the turnover rate is a product of the ß-VLDL fractional catabolic rate and the ß-VLDL pool size (first-order reaction). Thus, the ß-VLDL production rate equals the ß-VLDL fractional catabolic rate times ß-VLDL pool size. Since the chow-fed op0/E0 mice have a ß-VLDL pool size 2.5-fold larger and a ß-VLDL fractional catabolic rate 2.5-fold smaller than the op2/E0 mice, we can conclude that the ß-VLDL production rate need not be different to account for the observed difference in ß-VLDL pool size. Therefore, although the absolute mass of ß-VLDL cleared per hour is the same in the two different types of mice, the steady state levels of ß-VLDL are higher in the op0/E0 mice due to the decreased fractional catabolic rate. A similar result is obtained in LDL receptor deficiency states, in which the absolute mass clearance of LDL per hour, via receptor-independent pathways, is normal or even elevated due to the higher steady state levels of LDL caused by a decrease in its catabolic rate.⁵⁴ We do not know what receptor is responsible for the uptake of ß-VLDL in vivo, but the current study indicates that this receptor's activity or level is induced by M-CSF and most likely resides on monocytes/Ms. Recently, a specific and saturable receptor activity for ß-VLDL derived from apoE-deficient mice was characterized in a murine M cell line.⁵⁵ Binding of ß-VLDL to this receptor is not competed for by LDL or by AcLDL but is competed for by normal VLDL.⁵⁵

Recently, hepatic SR-A was overexpressed in transgenic mice to investigate the functional role of these site-specific receptors in clearing potentially atherogenic lipoproteins.⁵⁶ The mouse transferrin promoter targeted expression of the bovine SR-A type I to murine liver. Overexpression of hepatic SR-A enhanced cholesterol flux, and transgenic SR-A mice on an atherogenic diet accumulated less apoB-containing lipoprotein cholesterol and secreted more biliary cholesterol as bile acids. This increased removal of modified lipoproteins would be consistent with a protective antiatherogenic role for SR-A.

Indirect evidence for SR-A as a proatherogenic molecule emerged with the report that mice lacking tumor necrosis factor receptor p55 develop accelerated atherosclerosis.⁵⁷ The increase in lesion size was accompanied by a threefold elevation in SR-A activity and overexpression of SR-A in aortic sinus sections. Direct evidence was provided by the observation that targeted disruption of the SR-A gene results in a ±50% reduction in the size of atherosclerotic lesions in apoE-deficient mice.¹⁰

The explanation for the seeming paradox in the atherogenic effects of low and high SR-A expression may be the tissue location of expression and effects on cholesterol flux. In addition, the balance or competition that exists in vivo between arterial subendothelial SR-A and liver Kupffer and sinusoidal endothelial SR-A for removal of modified lipoproteins may prove of critical importance. Thus, when hepatic SR-A is overexpressed, modified lipoproteins would be less likely to bind to SR-A present on Ms in the aortic subendothelium (antiatherogenic scenario). ApoE-deficient mice, in contrast, develop hypercholesterolemia due to decreased hepatic clearance of atherogenic lipoproteins, thereby increasing access of modified lipoproteins to subendothelial Ms. This shifts the balance to increased SR-A binding, enhanced arterial lipid deposition, and foam cell formation (proatherogenic scenario). In op0/E0 mice, hepatic uptake and clearance of atherogenic lipoproteins may be inefficient due to reduced Kupffer cell numbers and SR-A expression, resulting in severe hypercholesterolemia. The reduction in M-CSF–dependent vascular tree M numbers (subendothelial Ms and myocardial interstitial Ms) and SR-A expression, however, limits arterial modified lipoprotein uptake, and severe atherosclerosis fails to develop.

This hypothesis would be consistent with several studies that have demonstrated M-CSF–induced decreases in plasma cholesterol in normal rabbits and hypercholesterolemic LDL receptor–defective Watanabe heritable hyperlipidemic rabbits, nonhuman primates, and normocholesterolemic and hypercholesterolemic humans, as well as patients with homozygous familial hypercholesterolemia.^{5 6 7 58} M-CSF also decreased foam cell development in M-rich carrageenan-induced granulomas in Watanabe heritable hyperlipidemic rabbits and decreased progression and enhanced regression of atherosclerotic lesions in Watanabe heritable hyperlipidemic and cholesterol-fed New Zealand White rabbits.^{7 8 58} The cholesterol-lowering effects of M-CSF have been ascribed to (1) enhanced biliary cholesterol excretion,⁵⁸ (2) the upregulation of apoE secretion by Ms, thereby enhancing M cholesterol efflux and reverse cholesterol transport,⁵⁹ and (3) increased expression of additional M receptors important in cholesterol clearance, such as the apoE-binding protein LDL receptor–related protein.^{60 61} In conclusion, we have characterized the M phenotype in op2/E0, op1/E0, and op0/E0 mice and have clearly illustrated the importance of M-CSF and M-CSF–dependent monocyte/M subpopulations in maintaining cholesterol homeostasis and in the pathogenesis of atherosclerosis.

	Selected Abbreviations and Acronyms

 

AcLDL = acetylated LDL apo = apolipoprotein EC = endothelial cell M = macrophage M-CSF = macrophage-colony–stimulating factor mAb = monoclonal antibody op = osteopetrotic SR-A = scavenger receptor

	Acknowledgments

 
This work was supported by the British Heart Foundation and UK Medical Research Council and Arthritis and Rheumatism Council (S. Gordon), Physician/Scientist Award, University of Kentucky (W.J.S. de Villiers), an Established Investigatorship from the American Heart Association, and grant PO1 HL54591 from the NIH (J.D. Smith). W.J.S. de Villiers is a Nuffield Dominion Medical Fellow at Wolfson College, Oxford. Recombinant cholesterol ester transfer protein was generously provided by Alan Tall, Columbia University.

Received February 17, 1997; accepted November 26, 1997.

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