1Department of Pathology, 2Division of Cardiovascular Medicine, Department of Medicine, and 3Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee
Submitted 28 April 2004 ; accepted in final form 19 October 2004
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ABSTRACT |
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autocrine/paracrine system; cholesterol acceptor; extracellular space; distribution volume
Making comparisons between the autocrine and paracrine effects of apoE synthesized by macrophages has proven difficult. For in vitro experiments with cultured macrophages, exogenously applied apoE has been used to approximate paracrine or endocrine apoE (14, 19). Endogenously synthesized apoE has been used to approximate autocrine apoE (16, 1921). These approximations do not consider that a single molecule of an endogenously synthesized mediator can have a series of interactions that employ a combination of autocrine and paracrine mechanisms (4).
The novel methodology discussed here was developed to separate autocrine and paracrine mechanisms by using a simple mathematical model to interpret mediator-induced biological effects. This study shows that whereas macrophage apoE has both autocrine and paracrine effects on cholesterol efflux, autocrine apoE has smaller but more consistent effects than paracrine apoE. Whereas macrophage apoE is used to demonstrate this novel methodology, the concepts have applications for separating the autocrine and paracrine effects of many other secreted biological mediators including growth factors, cytokines, and carrier proteins.
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Glossary |
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MATERIALS AND METHODS |
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Lipoprotein preparation and chemical modification. Lipoproteins were isolated from human blood plasma by sequential ultracentrifugation with density 1.0191.063 g/ml for LDL. LDL was dialyzed at 4°C in lipoprotein buffer (0.15 M NaCl and 0.3 mM EDTA). acLDL was prepared by repeated addition of acetic anhydride to LDL (2). After the modification reaction, the acLDL was dialyzed in lipoprotein buffer. Lipoprotein species and modifications were confirmed by electrophoretic mobility on agarose gels. Protein concentrations were determined by a modified Lowry assay (23). Modified lipoproteins were filtered with 0.45-µm syringe filters immediately before being applied to cultured cells.
Efflux of cellular cholesterol.
Macrophages were labeled with 2 µCi/ml [1,2-3H(N)]cholesterol (Perkin Elmer Life Sciences, Boston, MA) in DMEM for 12 h (19, 26). ApoE+/+ and apoE/ macrophages had 64.1 ± 5.0 and 98.0 ± 12.3 µg total cholesterol/mg cell protein, respectively. apoE+/+ and apoE/ macrophages had 786.66 ± 133.36 and 782.8 ± 98.93 cpm/mg cell protein, respectively. Labeled macrophages were rinsed three times with DMEM-0.2% BSA. The efflux period was initiated by the addition of DMEM with no acceptors (0.3, 0.5, 0.75, 1.0, 1.5, or 2.0 ml). Efflux medium was removed after an efflux period of 8, 24, 48, or 72 h, and cell debris was removed by centrifugation. Remaining cellular [3H]cholesterol was harvested by rinsing cells with PBS and then lysing cells with 1.0 ml of 0.1 N NaOH. Counts (cpm) in medium and lysate were detected with a Beckman LS 6000IC scintillation counter and Ecolite scintillation fluid (ICN, Costa Mesa, CA). Cholesterol efflux was calculated from the counts in the medium as a percentage of the total counts (medium + lysate). The difference in cholesterol efflux (E) between apoE+/+ (KO) and apoE/ (WT) macrophages is considered to be the cholesterol efflux that is specifically mediated by apoE. E is calculated as
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Western blot analysis of secreted apoE. Macrophages were incubated in DMEM with no acceptors (0.3, 0.5, 0.75, 1.0, 1.5, or 2.0 ml) for 18 h, and cell debris was removed by centrifugation. ApoE was extracted from the total volume of culture medium with Liposorb gel (Calbiochem, San Diego, CA). For Western blot analysis of secreted apoE, extracted apoE was separated by 412% NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose membranes. ApoE was detected with anti-murine apoE rabbit antiserum and visualized by chemiluminescent ECL Plus (Amersham Biosciences, Piscataway, NJ).
14C-labeled adenine release assay for cellular toxicity. Cytotoxicity in cultured macrophages was assayed by measuring the release of 14C-labeled adenine (34). Cells were labeled for 3 h in DMEM with 0.4 µCi/ml [U-14C]adenine (Amersham Biosciences). Cells were rinsed three times with DMEM. The release period was initiated by the addition of DMEM. Medium was removed after a release period of 24 h, and cell debris was removed by centrifugation. Cellular [14C]adenine was harvested by rinsing cells with PBS and then by lysing cells with 1.0 ml of 0.1 N NaOH. Sample aliquots were loaded into Ecolite scintillation fluid, and [14C]adenine counts were detected with a Beckman LS 6000IC scintillation counter. Adenine release was calculated from the medium [14C]adenine counts and expressed as a percentage of the total counts (lysate + medium).
Volume dependence theory.
To manipulate the extracellular concentration of an endogenously synthesized biological mediator, the volume dependence theory takes advantage of the implicit relationship between concentration and distribution volume. The concentration of a secreted mediator such as apoE is proportional to the reciprocal of the extracellular distribution volume. Increasing the distribution volume (V) causes a decrease in the relative concentration (V1) of the secreted mediator. Varying the extracellular volume of a given number of cells (Fig. 1A) allows the juxtacellular and extracellular mechanisms of the synthesized mediator to be mathematically separated and characterized. This theory can be illustrated graphically as follows. For a plot of the biological effect (E) vs. extracellular volume (V), as the extracellular volume is increased, the effect decreases asymptotically to a plateau that represents the portion of the total effect that is independent of the extracellular volume (Fig. 1B). The value of this plateau is the y-intercept (b) of a plot of the biological effect (
E) vs. reciprocal volume (V1) (Fig. 1C). This dose-response curve is linear at low relative concentrations (V1) and fits the linear equation:
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RESULTS |
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Model for apoE-mediated cholesterol efflux.
With a limited number of assumptions, a mathematical model can be generated that suggests a physical model for the autocrine and paracrine effects of apoE on cholesterol efflux. The calculation of E in Eq. 1 assumes that any difference is a primary effect of apoE and not a secondary effect of changes in cellular cholesterol homeostasis (i.e., changes in viability or changes in membrane fluidity). The data in Fig. 2, Table 1, and Fig. 3 assume that evaporation of the extracellular distribution volume is negligible. However, evaporation was determined to be
0.03 ml/24 h for this system. The evaporation rate affects the calculation of
E and the calculation of V1. Factoring in the evaporation rate does not change the trends in the data. However, the evaporation corrections are necessary to estimate the parameters in the mathematical model (data not shown).
From an extrapolation of the concentration dependence lines, a common x-intercept is observed and is assigned a value of negative C (0.270 ± 0.047 ml1) (Fig. 4A). The common x-intercept (C, 0) for all the lines that fit Eq. 2 yields the following equation:
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DISCUSSION |
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Our study suggests that autocrine apoE has advantages that are biologically relevant. Even when the extracellular space is saturated with apoE, at least 1020% of the apoE-mediated cholesterol efflux is the result of volume-independent interactions of endogenous apoE (Table 1). Lin et al. (19) found that with apoE-deficient cells, the exogenous application of apoE at 10 times the concentration of normal endogenous apoE secretion did not increase cholesterol efflux as much as normal endogenous apoE secretion by apoE-producing cells. The fundamental importance of these media transfer studies is that they show that exogenous apoE (paracrine) is not equivalent to endogenous apoE (autocrine and paracrine). For a macrophage with endogenously synthesized apoE, the advantage of autocrine apoE may become critical if homeostasis is challenged by cholesterol loading, if the conditioned space is large (making apoE diluted), if the turnover of conditioned space is high because of interstitial flow, or if a macrophage is isolated from other macrophages. The interstitial concentration, distribution volume, and turnover of macrophage apoE in the vessel wall are not known. These factors are critical for the interpretation of data from in vitro cell culture systems and the generalization of these data to the biology of macrophages in vivo. For example, the trend in viability may be related to changes in apoE secretion observed at very low volumes (Fig. 3A). The log-linear trend in viability (Fig. 3C) may be due to the relative concentrations of confounding mediators like growth factors and culture gases (e.g., CO2) in varying distribution volumes. Because apoE is not the only mediator being secreted in our system, it was necessary to subtract the effects of confounding mediators. This is why apoE+/+ macrophages were compared with apoE/ macrophages and why the specific apoE effect was calculated as apoE-mediated cholesterol efflux (E). The effects of confounding mediators on cholesterol efflux were minimal (Fig. 2, A and B). Although the major role of apoE is as an extracellular cholesterol acceptor, it is also possible that a portion of the effect of apoE on cholesterol efflux is the consequence of other actions of apoE, such as receptor or proteoglycan binding (17), intracellular cholesterol routing (8), or even stabilization of the cytoskeleton (6). Irrespective of which ultimate mechanisms or combination of effects may be responsible for the modulation of cholesterol efflux by apoE, these can only occur through either autocrine or paracrine events and are therefore addressed as a whole in our study.
Although the volume dependence of apoE secretion and viability could not explain the volume-dependent trends of apoE-mediated efflux (Fig. 2C), these data definitely underscore the sensitivity of biological processes to in vitro culture volumes. Some of the higher-order characteristics of macrophage tissue could be overlooked by in vitro studies with macrophages. For example, an aggregate of macrophages may become a tissue that works with autocrine efficiency instead of being just the sum of its autocrine/paracrine cellular units. The spatial range of autocrine effect (30) is very important because if another cell is within this range there is the possibility of autocrine cooperation. This possibility could be explored by in vitro or in vivo studies on the effects of the geometry and the density of cell aggregates on apoE-mediated cholesterol efflux. Compared with exogenous lipoprotein-bound apoE of hepatic origin, local secretion of lipid free apoE by macrophages results in decreased atherosclerosis due to a small amount of apoE in a critical location (12, 22). In the study by Fazio et al. (12), WT mice were transplanted with apoE/ bone marrow. These chimeric mice had normal plasma apoE but no macrophage apoE production in the vessel walls. Although immunocytochemical staining of lesions with WT macrophages reveals high apoE levels, these chimeric mice with only plasma apoE had little to no apoE in the lesions. The lack of staining for plasma-derived apoE within the artery wall suggests that penetration into clusters of macrophages is limited. The macrophage, not plasma-derived apoE, is the primary source of apoE for RCT (5, 12, 22). Along these same lines, paracrine apoE may not be able to fulfill the critical actions of the small amount of autocrine apoE in the juxtacellular space or possibly even the intracellular space. apoE recycling, the internalization and resecretion of apoE (10, 13, 33), has been reported to mediate cholesterol efflux in hepatocytes and in macrophages (13). The methodology in this study is unable to resolve the "internal autocrine" (31) or "intracrine" (28, 29) mechanism that could be mediated by intracellular apoE.
Proposed model.
We propose a model from Eq. 6 in which macrophages secrete apoE to create an extracellular sink for cellular cholesterol. The capacity, concentration, and compartmentalization of the sink affect cholesterol efflux (Fig. 5). The sink has a capacity (ft) that expands as apoE is secreted. The sink can be separated into two compartments that are a consequence of the location of apoE in juxtacellular or extracellular space, also described as the secretion layer and the bulk layer, respectively (18, 27). The dilution of apoE as it moves to the extracellular space diminishes its efficacy. ApoE-mediated efflux (E) is a balance between the efflux and influx of cholesterol. Efflux depends on the sink capacity (ft), the concentration of extracellular apoE (V1), and the functional concentration of juxtacellular apoE (C). Influx depends on the cholesterol in the apoE sink (
E) and the functional concentration of juxtacellular apoE (kI). Decreases in the functional concentration of apoE that contribute to influx (nt) may represent changes in the capacitance of apoE due to accumulation of phospholipids that stabilize cholesterol or to proteolytic alteration of apoE. It is also possible that n is an artifact of slight changes in viability or apoE secretion.
Autocrine/paracrine systems. In many of the documents that are found by a literature search for the term "autocrine," the terms "autocrine and/or paracrine" describe the proposed mechanism of action. It is difficult to quantitatively separate these two mechanisms experimentally. Traditional approaches to this problem have limitations and rely on simple qualitative assessments. Traditional methodologies that have been used to study macrophage apoE include characterizing the tissue distribution of apoE (11), measuring the effects of the endogenous synthesis of apoE vs. its complete absence (11), and measuring the effects of the endogenous synthesis vs. exogenous application of apoE (15, 19). These methods are useful in comparative assessments, but they are unable to simultaneously quantify the serial effects mediated by apoE or other secreted biological mediators.
In the present study, we have performed experiments based on the theory that the portion of the endogenous acceptor that mediates autocrine effects cannot be diluted. Previous studies have utilized experimental strategies similar to the one used in our study. Steck et al. (32) performed volume dependence experiments to study whether efflux to exogenous acceptors is proceeded by aqueous diffusion of cholesterol or by acceptor-membrane collisions. They found that, for certain types of acceptors, efflux approached a "volume-insensitive plateau," and they proposed that these types of acceptors may have "sites for efficient collisional transfer" (32). Our studies with endogenously synthesized acceptors suggest that these sites may be a combination of binding sites on the membrane and an enriched aqueous layer around the membrane (i.e., the juxtacellular space). Oehrtman et al. (27) used a theoretical model to study the escape of autocrine signaling ligands into the extracellular space. They found in a model of a ligand-receptor signaling system that "varying volume heights shows little effect on ligand concentrations." It remains to be seen whether the dilution methodology discussed here can change mediator concentrations over the large dynamic range (many orders of magnitude) of a log-linear dose-response curve such as those seen for ligand-receptor signaling pathways.
In summary, this study separates and quantifies the autocrine and paracrine effects of apoE on cholesterol efflux from macrophages. These data suggest that the relative importance of autocrine and paracrine apoE depends on the size of the local distribution volume, with the autocrine effects remaining constant even when the paracrine effects are diminished by the dilution of apoE.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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