Enhanced Upregulation of the Fc Receptor IIIa (CD16a) During In Vitro Differentiation of ApoE4/4 Monocytes

From the Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany.

Correspondence to Gerd Schmitz, MD, Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Franz-Josef-Strauß-Allee 11, D-93053 Regensburg, Germany. E-mail gerd.schmitz{at}klinik.uni-regensburg.de

	Abstract

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Abstract—We recently reported a positive correlation of the pool size of lipopolysaccharide receptor (CD14)^dim and Fc receptor IIIa (CD16a)⁺ monocytes in peripheral blood to the apolipoprotein E4 (apoE4) phenotype and a negative correlation to high density lipoprotein (HDL) cholesterol levels (Arterioscler Thromb Vasc Biol. 1996;16:1437–1447). In this study, the in vitro differentiation of mononuclear phagocytes derived from healthy blood donors homozygous for the E3/3 or the E4/4 phenotype was analyzed during 7 days of culture in serum-free medium supplemented with macrophage colony–stimulating factor (M-CSF). The CD16a expression, which indicates Fc receptor–dependent phagocytic activity, increased to a significantly higher level in apoE4/4 monocytes than in apoE3/3 cells. The costimulatory molecule CD40, which indicates antigen-presenting capacity, was upregulated more strongly in apoE3/3 monocytes compared with E4/4 cells, but the difference did not reach a significant level. The expression of differentiation-associated surface proteins (CD14, CD33, CD45) and adhesion molecules (CD11a, CD11b, CD11c, CD49d) was not significantly different between apoE3/3 and apoE4/4 monocytes. However, a significantly decreased intracellular apoE concentration and a reduced amount of secreted apoE were found in apoE4/4 monocytes during in vitro differentiation. No differences were found in the surface expression of the low density lipoprotein receptor–related protein (CD91) and the uptake of fluorescence labeled low density lipoprotein between apoE3/3 and apoE4/4 monocytes. These data indicate that the apoE4/4 phenotype significantly influences the M-CSF–dependent differentiation of monocytes toward a more CD16a-positive phagocytic phenotype.

Key Words: monocytes • apolipoprotein E • CD16a • CD40 • low density lipoprotein receptor–related protein

	Introduction

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Atherosclerosis is considered to represent a chronic inflammatory disease, and in atherosclerotic plaques other than lipid-laden foam cells and other cell types of monomyeloid origin, activated T cells also are detectable.¹ Furthermore, major characteristics of systemic inflammation such as enhanced plasma concentration of C-reactive protein,² fibrinogen, and complement components,³ as well as leukocytosis⁴ are associated with the progression of atherosclerosis. During this process, monocytes show an increased adhesion to injured endothelium that leads to enhanced migration of these cells into the vessel wall.¹ Within the vessel wall, monocytes transform to scavenger cells that take up excessive amounts of lipoprotein-derived lipids, a process that leads to foam cell formation and the development of fatty streaks.¹

In peripheral blood, an altered proportion of mononuclear phagocytes with a phenotype similar to alveolar macrophages is detectable,⁵ which has a high Fc receptor IIIa (CD16a) expression associated with IgG-dependent phagocytic activity. This CD14^dim subset is negatively correlated to HDL cholesterol levels⁶ but positively correlates to total plasma cholesterol (G.R. et al, unpublished data, 1998). These cells are further expanded in correlation to the apolipoprotein E4 (apoE4) allele⁶ and in genetic defects of lysosomal acid lipase⁷ and ß-galactosidase.⁸ Changes in the distribution of mononuclear phagocyte subsets have also been shown with other diseases, including cancer, acute and chronic inflammation, and HIV infection,⁹ as well as under macrophage colony–stimulating factor (M-CSF) and granulocyte macrophage colony–stimulating factor (GM-CSF) therapy.^{9 10} The proinflammatory cytokine pattern of the CD16⁺ monocytes,¹¹ characterized by a lower expression of interleukin-10 (IL-10) and an unchanged expression of tumor necrosis factor- (TNF-), may indicate a modulatory role of these cells in the inflammatory process.

ApoE is a major component of several plasma lipoproteins, including VLDL, IDL, chylomicron remnants, and certain subclasses of HDL and is synthesized in hepatocytes, cells of the central nervous system, macrophages, and various other cells.¹² During monocyte-to-macrophage differentiation, apoE mRNA expression as well as apoE synthesis are upregulated.¹³ This occurs due to both an increased transcription rate of the apoE gene as well as a prolonged lifetime of the apoE mRNA. In animal models, an important role of apoE in the development of atherosclerosis has been demonstrated.¹⁴ ApoE-deficient (apoE(-/-)) mice, created by gene targeting, are hypercholesterolemic and spontaneously develop atherosclerosis.¹⁴ Significantly smaller proximal aortic lesions were found during crossbreeding of apoE(-/-) with M-CSF–deficient (op) mice.¹⁵ Transplantation of normal bone marrow into apoE(-/-) mice also results in the prevention of hypercholesterolemia and aortic and coronary atherosclerosis.¹⁶ These data strongly indicate that apoE synthesized by bone marrow–derived cells associates with serum lipoproteins and accelerates their clearance in vivo.

In humans 3 major isoforms of apoE are designated E2 (Cys¹¹² and Cys¹⁵⁸), E3 (Cys¹¹² and Arg¹⁵⁸), and E4 (Arg¹¹² and Arg¹⁵⁸), which are products of 3 alleles at a single gene locus.¹⁷ The apoE4 phenotype has been shown to be a risk factor for atherosclerosis,¹⁸ and the pathogenic role of apoE4 in amyloidosis and in patients with sporadic¹⁹ and late-onset familial^{19 20} Alzheimer's disease is currently under intensive investigation. ApoE-containing lipoproteins are ligands for various receptors, including the apoB/E receptor, VLDL receptor, LDL receptor–related protein (LRP; CD91), and the recently described apoE receptor II, which is highly expressed in brain and placenta.²¹ The apoE4 isoform is characterized by a normal affinity to the apoB/E receptor when compared with the E3 form.²² In individuals homozygous for the E4 isoform independently of normal receptor affinity, increased total serum cholesterol and LDL levels are observed.¹⁸ Increased deposition of ß-amyloid (Aß) protein in the brain and more neurofibrillary tangles²³ and a reduced cerebral glucose metabolism²⁴ were reported for apoE4-carrying subjects. ApoE4 furthermore does not increase neurite outgrowth in vitro as shown for apoE3.²⁵ These findings indicate multiple cellular differences between E3/3 and E4/4 individuals; however, the precise mechanisms are not yet understood.

Here we present data indicating that the apoE4/4 phenotype is correlated with an altered M-CSF–dependent differentiation of monocytes based on a more CD16a-positive phenotype that allows IgG-dependent phagocytosis at the same time with an impaired apoE content and secretion.

	Methods

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Blood Samples
Heparinized and EDTA-anticoagulated peripheral blood samples for ex vivo analysis were obtained from healthy female and male volunteers (aged 22 to 37) with either apoE3/3 or E4/4 phenotype, recruited from the hospital personnel and university students. All probands were free of disease or infection based on physical examination. Informed consent and approval of the Hospital Ethics Committee were obtained.

Monocyte Isolation and Cultivation
Human peripheral blood monocytes from healthy normolipemic volunteers were isolated by leukapheresis, and subsequent to a density gradient centrifugation over Histopaque-1077, monocytes were purified by counterflow centrifugation as previously described.²⁶ Peripheral blood mononuclear cells were isolated by a density gradient centrifugation over Histopaque-1077. Isolated monocytes or mononuclear cells were cultured up to 7 days at a concentration of 5x10⁵ to 1x10⁶ cells/mL in a serum-free medium (Macrophage-SFM, Gibco Life Technologies), supplemented with 50 ng/mL human recombinant M-CSF expressed in yeast (Genzyme Diagnostics, Cambridge, Mass) in 20-cm² petriPERM dishes (Heraeus Instruments) with a hydrophobic Teflon bottom. The cells were harvested by smoothly scratching with a Sarstedt cell scraper (Newton, NC) after a 30-minute incubation on ice.

Staining for Cell Surface Immunofluorescence
For flow cytometric immunophenotyping, unseparated whole blood (100 µL) was incubated for 15 minutes on ice with saturating concentrations of the fluorochrome-conjugated antibodies. The monoclonal antibodies CD11a (clone 25.3.1), CD14 (RMO52), CD16a (3G8), CD33 (My9), and CD49d (HP2/1) were obtained as fluorescein isothiocyanate or R-phycoerythrin (R-PE) or R-phycoerythrin-cyanin 5 (PE-Cy5) conjugates from Coulter-Immunotech (Hamburg, Germany). CD40 (EA-5) was obtained as an R-PE conjugate from Ancell (Bayport, Minn). CD11b (Leu-15), CD11c (Leu-M5), CD45 (Hle-1), and CD56 (Leu-19) were obtained as R-PE or peridinin chlorophyll conjugates from Becton Dickinson (Heidelberg, Germany). CD91 anti-LRP monoclonal antibody (8G1) was obtained from Progen (Heidelberg, Germany) as a biotin conjugate. IgG₁ and IgG_2a isotype controls were obtained as fluorescein isothiocyanate, R-PE, and peridinin chlorophyll conjugates from Becton Dickinson. Lysing of erythrocytes and washing were performed as previously described.⁶ In cultured monocytes, 5x10⁵ cells were incubated in 100 µL PBS for 15 minutes on ice with saturating concentrations of antibodies and washed 2 times with PBS before measurement.

Staining for Intracellular Immunofluorescence
For the flow cytometric determination of intracellular apoE, 5x10⁵ cultured cells in 100 µL PBS were stained as previously described for CD68⁶ with a monoclonal mouse anti-human apoE antibody (clone IX8fR39d; from our own laboratory).

Cellular Uptake of LDL Labeled With 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate (DiI)
Human LDL (d=1.006 to 1.063 g/mL) was obtained by sequential density gradient ultracentrifugation from normal human plasma as previously described.²⁷ Cellular uptake of LDL labeled with DiI²⁸ was followed by incubation of mononuclear cells with DiI-LDL for 45 minutes at 37°C. At the end of the incubation, cells were analyzed for DiI fluorescence by flow cytometry.

Flow Cytometric Analysis
The flow cytometric analyses were performed as previously described.⁶ Subpopulations of mononuclear phagocytes were identified as shown in Figure 1. The population of CD14^brightCD16a^- cells was defined based on a bright expression of CD14 and lack of CD16a expression. The CD14^brightCD16a⁺ cells show a bright CD14 expression at the same time as CD16a expression. The CD14^dimCD16a⁺ population was defined based on positive staining for CD16a and only dim CD14 expression. A fourth monocyte subpopulation was characterized by a dim CD14 expression and no expression of CD16a and population 5 by bright CD14 expression and positive staining for CD56. The CD14^dimCD16^- population could be subdivided further based on either a high or low expression of CD33.

View larger version (32K): [in this window] [in a new window]   Figure 1. Characterization of mononuclear phagocyte subpopulations on the basis of coexpression patterns of CD14 and CD16a (A), CD14 and CD33 (B), and CD16a and CD45 (C). The subsets were defined as follows: CD14brightCD16a- (MNP 1), CD14brightCD16a+ (MNP 2), CD14dimCD16a+ (MNP 3), CD14dimCD16a- (MNP 4), CD14dimCD16a-CD33bright (MNP 4a), CD14dimCD16a-CD33dim (MNP 4b), and CD14dimCD56+ (MNP 5).

The cellular antigen densities were calculated under the assumption of only 1 cellular binding site for each monoclonal antibody on its target antigen based on a calibration with reference beads carrying a defined number of anti-mouse binding sites (Flow Cytometry Standards Corporation, San Juan, Puerto Rico).

Analysis of Lipids and Lipoproteins
Cholesterol and triglyceride concentrations were determined by enzymatic methods using reagents from Boehringer Mannheim. HDL cholesterol and LDL cholesterol were determined after precipitation with reagents from Immuno. ApoA-I, apoA-II, and apoB were determined by end point nephelometry using reagents from Behring. The apoE polymorphism was analyzed by restriction isotyping by gene amplification and cleavage with Hha1.

ApoE Quantitation
ApoE was measured by a sandwich-type ELISA. Microtiter plates were coated overnight with polyclonal anti-apoE antiserum from sheep (Immuno, Vienna, Austria) in PBS and blocked with 2% BSA for 2 hours at 37°C. Diluted plasma samples or culture medium was added, and apoE was allowed to bind to coating antibodies. After washing, the amount of apoE bound to the microtiter plates was determined with a monoclonal mouse anti-human apoE antibody (clone IX8fR39d) and horseradish peroxidase–labeled anti-mouse antibodies (Dianova, Hamburg, Germany).

Statistical Analysis
Results are presented as mean and SD in an assumed normal distribution. The significance of differences between the size of monocyte subpopulations or antigen densities in probands with different apoE phenotypes was determined using te t test for independent samples. All calculations were performed using the SPSS/PC+ package (SPSS).

	Results

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Lipids, Lipoproteins, and Apolipoproteins of Blood Donors
The analyzed parameters of lipid and lipoprotein metabolism (Table 1) did not differ between the apoE3/3 and apoE4/4 blood donors. The analyzed values for both groups match with the normal range of typical healthy control subjects.

Monocyte Subpopulations Ex Vivo
In Table 2, the absolute cell counts of erythrocytes, lymphocytes, neutrophils, monocytes, basophils, and eosinophils are shown. No differences in the absolute cell numbers could be detected between apoE3/3 and apoE4/4 blood donors. However, significant differences between apoE3/3 and apoE4/4 individuals were found for the pool-size distribution of monocyte subsets ex vivo (Table 3). An increased CD14^brightCD16a^- population was detectable in apoE4 homozygous donors (apoE3/3=41.4%, apoE4/4=68.0%; P=0.006). At the same time, the CD14^brightCD16a⁺ population was significantly decreased (apoE3/3=47.9%, apoE4/4=16.7%; P=0.002). As in our previous study, the CD14^dimCD16a⁺ population also showed a tendency toward an increased proportion in apoE4/4 donors, which did not reach significance.

In addition, no significant differences were detected for the more immature CD14^brightCD56⁺ monocyte population and the CD14^dimCD16a^- population, which also expresses more CD33 and CD40 and resembles cells with a high antigen-presenting capacity. As shown in Figure 1B, this CD14^dimCD16a^- population was further divided into a CD33^bright and CD33^dim subset, but no significant differences according to the apoE phenotype were detectable.

Antigen Densities on Mononuclear Phagocytes Ex Vivo
To further characterize the peripheral blood mononuclear phagocytes from apoE3/3 and apoE4/4 donors, the expression densities of the lipopolysaccharide receptor, the Fc R IIIa–, ß₂-, and ß₁-integrins, a costimulatory molecule, and activation- and differentiation-associated membrane molecules were analyzed ex vivo (Table 4). For the ß₂-integrins CD11a, CD11b, and CD11c and the ß₁-integrin CD49d, the lipopolysaccharide receptor CD14, the pan-myeloid antigen CD33, the costimulatory molecule CD40, the leukocyte common antigen CD45 and its isoform CD45RA, and the maturity marker CD56, no differences in the expression densities were found when comparing apoE3/3 and apoE4/4 cells.

For CD16a, in contrast to the other antigens, the epitope number in peripheral blood mononuclear phagocytes, which was 18 800±27 800/cell in apoE3/3 donors and 4400±2400/cell in apoE4/4 donors, was different, correlating to the smaller size of the CD14^brightCD16a⁺ monocyte subpopulation.

Monocyte Subpopulations During In Vitro Culture
As changes in the composition of peripheral blood monocyte subpopulations can be correlated to altered maturation or extravasation, the in vitro differentiation of isolated monocytes from apoE4/4 donors was compared with monocytes from apoE3/3 donors (Figure 2). The isolated monocytes were cultured in hydrophobic dishes for easy removal by incubation on ice to allow single-cell analysis by flow cytometry. To exclude the effects of lipoproteins and lipids from the culture medium, a serum-free culture system was used.

View larger version (55K): [in this window] [in a new window]   Figure 2. Mononuclear phagocyte subpopulations during in vitro culture. Elutriated monocytes were cultured in serum-free medium supplemented with 50 ng/mL human recombinant M-CSF in hydrophobic dishes. For characterization of monocyte subpopulations, see Figure 1. (A) CD14 versus CD16a dot plot (day 0). (B) CD14 versus CD16a dot plot (day 1). (C) CD33 versus CD14 dot plot (day 0). (D) CD33 versus CD14 dot plot (day 1).

When simultaneously staining for either CD16a and CD14 or CD33 and CD14, a shift in the distribution of the populations was already visible on day 1 (Figure 2), indicating a differentiation of cells in our in vitro model. As shown in Figure 2A and 2C, increased CD16a and CD14 expression was induced during the first day of monocyte maturation, whereas CD33 remained almost unchanged (Figure 2B and 2D). Cells with a phenotype of the major monocyte population CD14^brightCD16a^- decreased, and at the same time, CD14^brightCD16a⁺, CD14^dimCD16a⁺, and CD14^dimCD16a^- cells increased as an effect of macrophage differentiation. However, a reliable determination of population sizes was already difficult on day 3. Therefore, the differentiation of the cells was further analyzed based on the expression densities of maturation-associated and adhesion antigens.

Expression of Maturation-Associated Antigens During Monocyte Differentiation
To characterize monocyte in vitro differentiation in correlation to the apoE phenotype, the expression of CD14 and CD16a as well as that of CD33, CD40, CD45, and CD56 was observed during 7 days of culture (Figure 3). The kinetics of CD16a expression significantly differed between E3/3 and E4/4 cells during M-CSF–dependent cell differentiation. In apoE3/3 monocytes (Figure 3A), CD16a expression reached its peak on day 7 (440±201% of initial values). The apoE4/4 monocytes (Figure 3B), in contrast, showed a higher expression of CD16a (953±429% of initial values) on day 7 and reached the peak level already on day 3 (1190±417%; P=0.006). CD45 and CD14 increased during the 7 days of differentiation (CD45: apoE3/3=316±41%, apoE4/4=261±112%; CD14: apoE3/3=310±77%, apoE4/4=372±150%), but no differential expression was detectable between apoE3/3 and apoE4/4 monocytes. In contrast, CD40, which is expressed preferentially on antigen-presenting cells, showed an enhanced kinetics of its surface expression in E3/3 monocytes on days 3 to 5 (day 3: apoE3/3=709±271%, apoE4/4=459±129%; day 5: apoE3/3=897± 453%, apoE4/4=711± 215%) (Figure 3A and 3B); however, the differences between the groups did not reach significance.

View larger version (19K): [in this window] [in a new window]   Figure 3. Expression of differentiation-associated antigens during 7-day monocyte culture. Expression densities were calculated for all cultured mononuclear phagocytes. Dead cells were excluded by propidium iodide staining. Values are given as percentage of freshly isolated monocytes (this expression density was set to 100%). Mean±SD values of 4 subjects for each apoE3/3 (A) and E4/4 (B) are shown. *P<0.05; **P<0.01.

Adhesion Antigen Expression During Monocyte Differentiation
Integrin expression densities were followed during in vitro monocyte differentiation to address whether adhesion antigen expression is influenced by the apoE phenotype; the results are shown in Figure 4A and 4B. For the integrins CD11a and CD11b, only small changes of expression were observed during 7-day differentiation (CD11a: apoE3/3=151±19%, apoE4/4=119±37%; CD11b: apoE3/3=149±50%, apoE4/4=144±61%). Higher increases during all 7 days of culture were observed for the ß₁-integrin CD49d (apoE3/3=311±102%, E4/4=379±171%) and the ß₂-integrin CD11c (apoE3/3=649± 230%, apoE4/4=564± 155%). ApoE4/4 in comparison with apoE3/3 monocytes showed a tendency toward a lower expression of CD11a and CD11c during the later part of the analysis period, which, however, with an exception in the case of CD11a on day 5 (P=0.040), did not reach significance.

View larger version (19K): [in this window] [in a new window]   Figure 4. Expression densities of integrins during 7-day monocyte culture. Expression densities were calculated for all cultured mononuclear phagocytes. Dead cells were excluded by propidium iodide staining. Values are given as percentage of freshly isolated monocytes (this expression density was set to 100%). Mean±SD values of 4 subjects for each apoE3/3 (A) and E4/4 (B) are shown. *P<0.05.

Intracellular ApoE Content and ApoE Secretion During Monocyte Differentiation
Mononuclear phagocytes are an important extrahepatic source of apoE in the human organism, and the apoE expression in monocytes is upregulated during cellular differentiation. Therefore, cellular apoE expression was analyzed intracellularly by flow cytometry using a monoclonal mouse anti-human apoE antibody in fixed and permeabilized cultured monocytes; the results are shown in Figure 5. In freshly isolated cells and during the first 3 days of monocyte differentiation, no apoE was detectable. On day 5, with 1 exception, only minor amounts of apoE were found in cultured monocytes. Seven-day differentiated monocytes from apoE3/3 donors (Figure 5A) showed a significantly higher intracellular pool of apoE than monocytes from apoE4/4 donors (P=0.016) (Figure 5B).

View larger version (24K): [in this window] [in a new window]   Figure 5. Intracellular and secreted apoE during monocyte differentiation. Mononuclear cells were isolated by density gradient centrifugation and cultivated as described in Methods. (A, B) Flow cytometric analysis of intracellular apoE using a monoclonal mouse anti-human apoE antibody by indirect immunofluorescence in permeabilized cells, (A) apoE3/3, (B) apoE4/4. (C, D) ApoE content of the culture medium was determined by ELISA with a polyclonal coating antibody and a monoclonal detection antibody as described in Methods, (C) E3/3, (D) E4/4. The thin lines show the results of double determinations of intracellular apoE content for each proband, and in every single analysis in secreted apoE, the bold lines represent the mean values. *P<0.05 for difference between E3/3 and E4/4.

Because this effect may be due to altered amounts of apoE secretion or changes in receptor-mediated processing due to differences in the affinity of apoE3 and E4, the amount of extracellular apoE was analyzed in the culture medium using an apoE ELISA. Because no exogenous apoE was added to our synthetic culture medium, the apoE concentration in the medium was correlated to the amount of secreted apoE from the cultured monocytes. On the first day of culture, the apoE concentration in the media was below the detection sensitivity of 20 µg/L. On the third day, detectable amounts were found in 3 of the 4 apoE3/3 samples (Figure 5C) and in 1 of the 4 apoE4/4 samples (Figure 5D). During further differentiation, the apoE concentration in all media was found to be increased on day 5 (apoE3/3=15 µg/L to 203 µg/L, apoE4/4=24 µg/L to 92 µg/L) and day 7 (apoE3/3=96 µg/L to 729 µg/L, apoE4/4=72 µg/L to 164 µg/L). On day 7, significant lower amounts of secreted apoE (P=0.028) were detectable in apoE4/4 monocytes (Figure 5D). These results are correlated to the expanded CD16a⁺ immunophenotype of monocytes in E4/4 individuals and to the altered apoE cell content in this phenotype.

LRP Expression and ApoB/E Receptor Activity During Monocyte Differentiation
The expression of LRP (CD91) and the uptake of DiI-LDL were analyzed during monocyte differentiation to address how CD91 expression and apoB/E receptor activity are correlated to monocyte maturation. The apoE phenotype and the results are shown in Figure 6. Mononuclear cells were isolated over Histopaque gradient centrifugation, and the CD91 expression of the monocytes and DiI-LDL uptake were analyzed during 7-day in vitro differentiation. The surface expression of LRP continuously increased (Figure 6A) and reached a maximum on day 5 (apoE3/3=510±161%, apoE4/4=512±108%). On day 7, the expression density was slightly decreased (apoE3/3=463± 203%, apoE4/4=403±142%). The similar results with cells from both groups of probands indicate that the alterations in apoE content and secretion in apoE4/4 monocytes are not associated with an altered surface expression of the CD91 molecule. In addition, the uptake of DiI-LDL (Figure 6B) as a measure of apoB/E receptor activity increased during M-CSF–dependent differentiation and reached a maximum on day 3 (apoE3/3=1449±772%, apoE4/4=997±175%) and then decreased during further differentiation until day 7 (apoE3/3=662±280%, apoE4/4=570±175%) without a difference between the groups of probands.

View larger version (17K): [in this window] [in a new window]   Figure 6. Expression density of LRP (A) and apoB/E receptor activity (B) during monocyte differentiation. Mononuclear cells were isolated by density gradient centrifugation and cultured as described in Methods. Values represent mean±SD of 4 subjects for each apoE3/3 and apoE4/4.

	Discussion

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In a previous study,⁶ we demonstrated a positive correlation of the pool size of CD14^dimCD16⁺ monocytes to the apoE4 allele and a negative correlation to HDL cholesterol. These cells, furthermore, were positively correlated to total plasma cholesterol (G.R. et al, unpublished data, 1998) and genetic defects of lysosomal acid lipase⁷ and lysosomal ß-galactosidase.⁸ This correlation of CD16a-expressing and CD14^dim monocytes to risk factors for atherosclerosis suggested a systemic immunological abnormality that might be an effect of either altered maturation or altered extravasation of mononuclear phagocytes into the vessel wall. In this study, we therefore analyzed the effects of the apoE4 phenotype on the in vitro differentiation of monocytes in more detail. In a first step, peripheral blood monocytes from healthy apoE3/3 and apoE4/4 subjects were compared in detail according to their subpopulation sizes and antigen profiles ex vivo, revealing an increase of the major CD14^brightCD16a^- population and the CD14^dimCD16a⁺ population in apoE4/4 subjects (Table 3). At the same time, the CD14^brightCD16a⁺ population that is similar in phenotype to more mature macrophages⁵ was decreased. These results confirm our previous results in the analysis of both healthy probands and hypercholesterolemic patients⁶ and correspond to a monocyte subpopulation pattern similar to that found in patients with inflammatory diseases.⁹

An increased proportion of more mature monocytes in subjects with the apoE4/4 phenotype could be due to either a higher plasma cholesterol concentration or an altered endogenous differentiation or extravasation behavior of the apoE4/4 monocytes, although no differences in plasma lipids were observed in this study (Table 1). Therefore, we further analyzed the in vitro differentiation of monocytes from apoE4/4 and apoE3/3 individuals. To exclude the influence of exogenous lipoproteins or cytokines, a serum-free culture medium was used for monocyte culture. Supplementation with recombinant human M-CSF supported the gradual differentiation of cells during the 7 days of analysis. Because a reliable determination of monocyte subpopulations was only possible for up to 3 days, the analysis was based on the expression densities of antigens on the total monocytic cell population. In this culture model, during in vitro differentiation, CD16a expression already increased earlier in apoE4/4-derived monocytes and, furthermore, reached higher expression densities than in E3/3 cells (Figure 3). As expected for a differentiation toward phagocytically active macrophages, there also was a tendency toward a lower increase in the expression density of the costimulatory molecule CD40 in apoE4/4 monocytes.

Comparable changes in the CD16a expression of peripheral blood mononuclear phagocytes, which in peripheral blood kinetically are associated with decreased CD14 expression, are not only found in association with atherogenic risk factors but also appear under conditions of enhanced host defense in inflammation and cancer.⁹ These CD14^dimCD16a⁺ monocytes that are phenotypically similar to alveolar macrophages⁵ seem to represent a more mature phenotype as suggested by a lower expression of the myelomonocytic stem cell marker CD33⁵ and a higher expression of the tyrosine kinase CD45.⁶ The lower capacity for production of the anti-inflammatory cytokine IL-10 and unchanged expression of TNF-¹¹ suggest that an increase of this CD14^dimCD16a⁺ monocyte population will further drive the inflammatory process.

Studies with different cytokines in vivo and in vitro have indicated potential mediators that may induce this altered composition of peripheral blood monocytes. Thus, therapy with M-CSF promotes the formation of CD16a⁺ cells,⁹ whereas GM-CSF treatment decreases the number of CD16a⁺ monocytes.¹⁰ In vitro IL-10 and transforming growth factor-ß (TGF-ß) induce CD16a expression,^{29 30} whereas GM-CSF³¹ or IL-4³² antagonize this effect. Similarly, GM-CSF and IL-4 cooperate in the induction of differentiation of peripheral blood monocytes toward dendritic cells, whereas IL-10 counteracts dendritic differentiation,³³ suggesting that the cytokine-dependent upregulation or downregulation of CD16a expression may be correlated to the cellular differentiation toward either a phagocytically active macrophage or dendritic cell development.

ApoE has been found accumulated both in atherosclerotic and senile plaques³⁴ and the apoE4/4 phenotype, which is associated with an enhanced cardiovascular risk^{17 18} and also with increased risk for early- and late-onset Alzheimer's disease, experimentally related to tight binding to Aß protein.^{19 20 35} Activation of the classic complement pathway appears to be a key factor in atherosclerosis³⁶ as well as in the progression of the chronic inflammatory response of Alzheimer's disease.^{37 38} Thus, Aß activates the classic complement pathway in vitro.³⁹ Further in vitro studies revealed an apoE4/4-specific enhancement of complement activation by Aß that may be related to its role in increasing the risk of early- and late-onset Alzheimer's disease,⁴⁰ and it is suspected that apoE modulates the beneficial role of soluble ß-amyloid precursor protein (sßAPP) to neural viability and the retention of partial Aß at the sßAPP carboxy terminus.⁴¹ ApoE and sßAPP functionally interact at the receptor level, and sßAPP containing the Kunitz protease inhibitor domain binds to LRP.⁴² Alternatively, heteromeric complexes of apoE, sßAPP, complement, and complement-associated IgG may render ligands for the CD16a/CR3/CR4 pathway.⁸

Because apoE itself is upregulated during monocyte differentiation,¹³ concomitantly with CD16a and scavenger receptors⁴³ we analyzed this lipoprotein in our culture system. After comparing the monocytes from E3/3 and E4/4 probands, a significantly lower intracellular and extracellular apoE concentration was found in E4/4-derived macrophages. These results agree with the previously described lower apoE concentration in apoE4/4 blood monocytes and the increased catabolism of apoE in apoE4/4 monocytes.⁴⁴ The cellular apoE concentration is regulated by cellular differentiation and is further affected by M-CSF that has been shown to increase apoE⁴⁵ in monocytes similar to CD16a.^{9 10} In mouse peritoneal macrophages, a similar increase of apoE was shown for TGF-ß and IL-1ß.⁴⁶ Interferon , in contrast, reduces apoE production concomitantly with the expression of LRP (CD91).⁴⁷

The multiligand receptor LRP (CD91) mediates the cellular uptake of apoE-containing lipoproteins, the ßAPP and various serine-proteinase/proteinase inhibitor complexes with ₂-macroglobulin, ₁-proteinase inhibitor, and plasminogen activator inhibitor.^{42 43 48} During monocyte differentiation, LRP expression is upregulated comparable to other scavenger receptors (eg, SRA I, II) or Fc receptors, a process that is promoted by M-CSF.⁴³ For apoE, an LRP-mediated regulatory loop was postulated in which cellular uptake of apoE through LRP enhances the synthesis and secretion of apoE. This would suggest a common regulation of apoE and LRP. However, in our experiments, in the presence of M-CSF, the LRP (CD91) expression increased in both apoE3/3 and apoE4/4 monocytes during differentiation, but no difference was found between the apoE3/3 and apoE4/4 cells. The same results were obtained with the apoB/E receptor activity as determined by the uptake of DiI-LDL. Because the affinity to the LDL receptor of apoE3- or apoE4-containing lipoproteins is not different,²² these results might suggest that the lower intracellular apoE concentration and the decreased secretion rate of apoE, which is correlated to the previously observed faster degradation,⁴⁴ are due to an increased uptake through a further lipoprotein receptor. Thus, the enhanced CD16a expression may be indicative for an upregulated regulatory loop of a scavenging principle that recognizes apoE4/4-sßAPP-complement-IgG complexes more efficiently than its apoE3/3 counterparts. The increased sequestration of endogenously synthesized apoE in the presence of lipoprotein lipase, however, suggests further potentially apoE4/4-sensitive internalization mechanisms.⁴⁹ Finally, also differential autocrine regulation of apoE and CD16a expression through the large variety of monocytic cytokines cannot currently be excluded. Further experiments should be performed to investigate the precise mechanisms of the selective upregulation of CD16a in apoE4/4 subjects and prove whether the apoE complexes in the medium contain sßAPP, complement, or IgG that would create a ligand complex for CD16a binding. This may lead to further insights into the development of atherosclerosis and Alzheimer's disease in subjects with the apoE4/4 phenotype.

Received January 23, 1998; accepted March 24, 1998.

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