Formation of ceramide-enriched domains in lipid particles enhances the binding of apolipoprotein E

We investigated the interaction between apolipoprotein E (apoE) and ceramide (CER)-enriched domains on the particles, by using lipid emulsions containing sphingomyelin (SM) or CER as model particles of lipoproteins. The sphingomyelinase (SMase)-induced aggregation of emulsion particles was prevented by apoE. CER increased the amount of apoE bound to emulsion particles. The confocal images of CER-containing large emulsions with two fluorescent probes showed three-dimensional microdomains enriched in CER. SMase also induced the formation of CER-enriched domains. We propose apoE prefers to bind on CER-enriched domains exposed on particle surface, and thus inhibits the aggregation or fusion of the particles.     

Macrophage cholesterol removal by triglyceride-phospholipid emulsions

Phospholipid liposomes were previously shown to mobilize cholesterol from cultured macrophage foam cells. Because Intralipid, a clinically available triglyceride-phospholipid emulsion, contains both phospholipid liposomes and triglyceride-emulsion particles, we sought to study its effect on macrophage cholesterol mobilization. Following an 18h incubation of J774 macrophages in serum-free medium supplemented with Intralipid, cholesteryl ester content decreased by up to 50% in previously cholesterol-loaded cells, and by 25% in non-loaded cells. Both components of Intralipid, liposomes and emulsion particles, independently caused reductions in cellular cholesteryl ester. We conclude that clinically available triglyceride-phospholipid emulsions can mobilize macrophage cholesterol in vitro.     

Effects of cholesterol in chylomicron remnant models of lipid emulsions on apoE-mediated uptake and cytotoxicity of macrophages

Chylomicron remnants have been suggested to be involved in the development of atherosclerosis. To investigate the mechanisms of chylomicron remnant-induced atherosclerosis, we prepared cholesterol (Chol)-containing emulsion particles as models for chylomicron remnants. Chol markedly increased the apolipoprotein E (apoE) binding maximum of emulsions without changing the binding affinity and thereby promoted emulsion uptake by J774 macrophages. Fluorescence measurements showed that Chol increased acyl chain order and head group hydration of the surface phospholipid (PL) layer of emulsions. The binding maximum of apoE was closely correlated with the hydration and the increase in the PL head group separation at the emulsion surface. From experiments using inhibitors for lipoprotein receptors, heparan sulfate proteoglycans and low density lipoprotein receptor-related protein were found to be the major contributors to the uptake of Chol-containing emulsions. Trypan blue dye exclusion revealed that the uptake of Chol-containing emulsions induced cytotoxicity to J774 macrophages. This study proposes a mechanism of atherosclerosis induced by chylomicron remnants.     

Uptake of type IV hypertriglyceridemic VLDL by cultured macrophages is enhanced by interferon-gamma.

Hypertriglyceridemic (HTG) very low density lipoproteins (VLDL) from subjects with type IV hyperlipoproteinemia induce both cholesteryl ester (CE) and triglyceride (TG) accumulation in cultured J774 macrophages. We examined whether the cytokine interferon-gamma (IFN-gamma), which is expressed by lymphocytes in atherosclerotic lesions, would modulate macrophage uptake of HTG -VLDL. Incubation of cells with HTG -VLDL alone significantly increased cellular CE and TG mass 17- and 4.3-fold, respectively, while cellular free cholesterol (FC) was unaffected. Pre-incubation of cells with IFN-gamma (50 U/ml) prior to incubation with HTG -VLDL caused a marked enhancement in cellular CE and TG 27- and 6-fold over no additions (controls), respectively, and a 1.5-fold increase in FC. IFN-gamma increased low density lipoprotein (LDL)-induced cellular CE 2-fold compared to LDL alone. IFN-gamma did not enhance the uptake of type III (apoE2/E2) HTG -VLDL or VLDL from apoE knock-out mice. Incubations in the presence of a lipoprotein lipase (LPL) inhibitor or an acylCoA:cholesterol acyltransferase (ACAT) inhibitor demonstrated that the IFN-gamma-enhanced HTG -VLDL uptake was dependent on LPL and ACAT activities. IFN-gamma significantly increased the binding and degradation of 125I-labeled LDL. Binding studies with 125I-labeled alpha2-macroglobulin, a known LDL receptor-related protein (LRP) ligand, and experiments with copper-oxidized LDL indicated that the IFN-gamma-enhanced uptake was not due to increased expression of the LRP or scavenger receptors. Thus, IFN-gamma may promote foam cell formation by accelerating macrophage uptake of native lipoproteins. IFN-gamma-stimulated CE accumulation in the presence of HTG -VLDL occurs via a process that requires receptor binding-competent apoE and active LPL. IFN-gamma-enhanced uptake of both HTG -VLDL and LDL is mediated by the LDL-receptor and requires ACAT-mediated cholesterol esterification.     

LDL Receptor-Related Protein-1 (LRP1) Regulates Cholesterol Accumulation in Macrophages

Within the circulation, cholesterol is transported by lipoprotein particles and is taken up by cells when these particles associate with cellular receptors. In macrophages, excessive lipoprotein particle uptake leads to foam cell formation, which is an early event in the development of atherosclerosis. Currently, mechanisms responsible for foam cell formation are incompletely understood. To date, several macrophage receptors have been identified that contribute to the uptake of modified forms of lipoproteins leading to foam cell formation, but the contribution of the LDL receptor-related protein 1 (LRP1) to this process is not known. To investigate the role of LRP1 in cholesterol accumulation in macrophages, we generated mice with a selective deletion of LRP1 in macrophages on an LDL receptor (LDLR)-deficient background (macLRP1-/-). After feeding mice a high fat diet for 11 weeks, peritoneal macrophages isolated from Lrp ^+/+ mice contained significantly higher levels of total cholesterol than those from macLRP1-/- mice. Further analysis revealed that this was due to increased levels of cholesterol esters. Interestingly, macLRP1-/- mice displayed elevated plasma cholesterol and triglyceride levels resulting from accumulation of large, triglyceride-rich lipoprotein particles in the circulation. This increase did not result from an increase in hepatic VLDL biosynthesis, but rather results from a defect in catabolism of triglyceride-rich lipoprotein particles in macLRP1-/- mice. These studies reveal an important in vivo contribution of macrophage LRP1 to cholesterol homeostasis.     

Lipoprotein aggregation protects human monocyte-derived macrophages from OxLDL-induced cytotoxicity

Oxidative modifications render low density lipoprotein cytotoxic and enhance its propensity to aggregate and fuse into particles similar to those found in atherosclerotic lesions. We showed previously that aggregation of oxidized LDL (OxLDL) promotes the transformation of human macrophages into lipid-laden foam cells (Asmis, R., and J. Jelk. 2000. Large variations in human foam cell formation in individuals. A fully autologous in vitro assay based on the quantitative analysis of cellular neutral lipids. Atherosclerosis. 148: 243-253). Here, we tested the hypothesis that aggregation of OxLDL enhances its clearance by human macrophages and thus may protect macrophages from OxLDL-induced cytotoxicity. We found that increased aggregation of OxLDL correlated with decreased macrophage injury. Using 3H-labeled and Alexa546-labeled OxLDL, we found that aggregation enhanced OxLDL uptake and increased cholesteryl ester accumulation but did not alter free cholesterol levels in macrophages. Acetylated LDL was a potent competitor of aggregated oxidized LDL (AggOxLDL) uptake, suggesting that scavenger receptor A plays an important role in the clearance of AggOxLDL. Inhibitors of actin polymerization, cytochalasin B, cytochalasin D, and latrunculin A, also prevented AggOxLDL uptake and restored OxLDL-induced cytotoxicity. This suggests that OxLDL-induced macrophage injury does not require OxLDL uptake and may occur on the cell surface. Our data demonstrate that aggregation of cytotoxic OxLDL enhances its clearance by macrophages without damage to the cells, thus allowing macrophages to avoid OxLDL-induced cell injury.     

Inhibition of macrophage inflammatory cytokine secretion by chylomicron remnants is dependent on their uptake by the low density lipoprotein receptor

Secretion of pro-inflammatory chemokines and cytokines by macrophages is a contributory factor in the pathogenesis of atherosclerosis. In this study, the effects of chylomicron remnants (CMR), the lipoproteins which transport dietary fat in the blood, on the production of pro-inflammatory chemokine and cytokine secretion by macrophages was investigated using CMR-like particles (CRLPs) together with THP-1 macrophages or primary human macrophages (HMDM). Incubation of CRLPs or oxidized CRLPs (oxCRLPs) with HMDM or THP-1 macrophages for up to 24h led to a marked decrease in the secretion of the pro-inflammatory chemokine monocyte chemoattractant protein-1 (MCP-1) and the pro-inflammatory cytokines tumour necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β (-50-90%), but these effects were reduced or abolished when CRLPs protected from oxidation by incorporation of the antioxidant drug, probucol, (pCRLPs) were used. In macrophages transfected with siRNA targeted to the low density lipoprotein receptor (LDLr), neither CRLPs nor pCRLPs had any significant effect on chemokine/cytokine secretion, but in cells transfected with siRNA targeted to the LDLr-related protein 1 (LRP1) both types of particles inhibited secretion to a similar extent to that observed with CRLPs in mock transfected cells. These findings demonstrate that macrophage pro-inflammatory chemokine/cytokine secretion is down-regulated by CMR, and that these effects are positively related to the lipoprotein oxidative state. Furthermore, uptake via the LDLr is required for the down-regulation, while uptake via LRP1 does not bring about this effect. Thus, the receptor-mediated route of uptake of CMR plays a crucial role in modulating their effects on inflammatory processes in macrophages.     

The effect of estrogens and phytoestrogenic lignans on macrophage uptake of atherogenic lipoproteins

The present study examined the effect of estrogens and compounds with estrogenic activity on the uptake of atherogenic lipoproteins into macrophages, thought to be the initiating step in the development of atherosclerotic lesions. Isolated low density lipoprotein (LDL) and lipoprotein(a) (Lp(a)) were radiolabelled with (3)H-cholesterol linoleate, and incubated with J774 macrophages for 24 hours in the presence of pharmacological doses of estrogens and phytoestrogens. At a concentration of 0.1 microM, the estrogen 17beta-estradiol significantly reduced LDL uptake by macrophages by 14% (p < 0.05), but estrone did not have any effect. At 10 microM, both estrogens significantly reduced macrophage LDL uptake, but the phytoestrogenic-lignans enterodiol and enterolactone had no effect on LDL uptake. Lp(a) uptake into cells was significantly reduced by both estrone and estradiol, and by enterolactone and enterodiol at concentrations of 10 microM (p < 0.01), with enterodiol being most effective. The results of this study suggest that the uptake of these structurally similar lipoproteins is regulated differently. Macrophage Lp(a) uptake appears more phytoestrogen sensitive than does LDL uptake.     

Atherogenicity of amino acids in the lipid-laden macrophage model system in vitro and in atherosclerotic mice: a key role for triglyceride metabolism

Atherosclerosis-related research has focused mainly on the effects of lipids on macrophage foam cell formation and atherogenesis, whereas the role of amino acids (AAs) was understudied. The current study aimed to identify anti- or pro-atherogenic AA in the macrophage model system and to elucidate the underlying metabolic and molecular mechanisms. J774A.1 cultured macrophages were treated with increasing concentrations of each 1 of the 20 AAs. Macrophage atherogenicity was assessed in terms of cellular toxicity, generation of reactive oxygen species (ROS) and cellular cholesterol or triglyceride content. At nontoxic concentrations (up to 1 mM), modest effects on ROS generation or cholesterol content were noted, but six specific AAs significantly affected macrophage triglyceride content. Glycine, cysteine, alanine and leucine significantly decreased macrophage triglyceride content (by 24%–38%), through attenuated uptake of triglyceride-rich very low-density lipoprotein (VLDL) by macrophages. In contrast, glutamate and glutamine caused a marked triglyceride accumulation in macrophages (by 107% and 129%, respectively), via a diacylglycerol acyltransferase-1 (DGAT1)-dependent increase in triglyceride biosynthesis rate with a concurrent maturation of the sterol regulatory element-binding protein-1 (SREBP1). Supplementation of apolipoprotein E-deficient (apoE^−/−) mice with glycine for 40 days significantly decreased the triglyceride levels in serum and in peritoneal macrophages (MPMs) isolated from the mice (by 19%). In contrast, glutamine supplementation significantly increased MPM ROS generation and the accumulation of cholesterol and that of triglycerides (by 48%), via enhanced uptake of LDL and VLDL. Altogether, the present findings reveal some novel roles for specific AA in macrophage atherogenicity, mainly through modulation of cellular triglyceride metabolism.     

Transport of beta-very low density lipoproteins and chylomicron remnants by macrophages is mediated by the low density lipoprotein receptor pathway

The receptor-mediated uptake of rat hypercholesterolemic very low density lipoproteins (beta VLDL) and rat chylomicron remnants was studied in monolayer cultures of the J774 and P388D1 macrophage cell lines and in primary cultures of mouse peritoneal macrophages. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was reduced 80-90% in the presence of high concentrations of unlabeled human low density lipoproteins (LDL). Human acetyl-LDL did not significantly compete at any concentration tested. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was also competitively inhibited by specific polyclonal antibodies directed against the estrogen-induced LDL receptor of rat liver. Incubation in the presence of anti-LDL receptor IgG, but not nonimmune IgG, reduced specific uptake greater than 80%. Anti-LDL receptor IgG, 125I-beta VLDL, and 125I-chylomicron remnants bound to two protein components of apparent molecular weights 125,000 and 111,000 on nitrocellulose blots of detergent-solubilized macrophage membranes. Between 70-90% of 125I-lipoprotein binding was confined to the 125,000-Da peptide. Binding of 125I-beta VLDL and 125I-chylomicron remnants to these proteins was competitively inhibited by anti-LDL receptor antibodies. Comparison of anti-LDL receptor IgG immunoblot profiles of detergent-solubilized membranes from mouse macrophages, fibroblasts, and liver, and normal and estrogen-induced rat liver demonstrated that the immunoreactive LDL receptor of mouse cells is of a lower molecular weight than that of rat liver. Incubation of J774 cells with 1.0 micrograms of 25-hydroxycholesterol/ml plus 20 micrograms of cholesterol/ml for 48 h decreased 125I-beta VLDL uptake and immuno- and ligand blotting to the 125,000- and 111,000-Da peptides by only 25%. Taken together, these data demonstrate that uptake of beta VLDL and chylomicron remnants by macrophages is mediated by an LDL receptor that is immunologically related to the LDL receptor of rat liver.     

Enhanced vascular permeability facilitates entry of plasma HDL and promotes macrophage-reverse cholesterol transport from skin in mice

Reverse cholesterol transport (RCT) pathway from macrophage foam cells initiates when HDL particles cross the endothelium, enter the interstitial fluid, and induce cholesterol efflux from these cells. We injected [³H]cholesterol-loaded J774 macrophages into the dorsal skin of mice and measured the transfer of macrophage-derived [³H]cholesterol to feces [macrophage-RCT (m-RCT)]. Injection of histamine to the macrophage injection site increased locally vascular permeability, enhanced influx of intravenously administered HDL, and stimulated m-RCT from the histamine-treated site. The stimulatory effect of histamine on m-RCT was abolished by prior administration of histamine H1 receptor (H1R) antagonist pyrilamine, indicating that the histamine effect was H1R-dependent. Subcutaneous administration of two other vasoactive mediators, serotonin or bradykinin, and activation of skin mast cells to secrete histamine and other vasoactive compounds also stimulated m-RCT. None of the studied vasoactive mediators affected serum HDL levels or the cholesterol-releasing ability of J774 macrophages in culture, indicating that acceleration of m-RCT was solely due to increased availability of cholesterol acceptors in skin. We conclude that disruption of the endothelial barrier by vasoactive compounds enhances the passage of HDL into interstitial fluid and increases the rate of RCT from peripheral macrophage foam cells, which reveals a novel tissue cholesterol-regulating function of these compounds.     

Uptake of modified low-density lipoproteins alters actin distribution and locomotor forces in macrophages

It is postulated thatmacrophage-derived foam cells accumulate in the arterial wall becausethey lose the ability to migrate after excessive ingestion of modifiedforms of low-density lipoproteins (LDL). To assess changes in locomotorforce generating capacity of foam cells, we measured isometric forcesin J774A.1 macrophages after cholesterol loading with oxidized (Ox-LDL)or aggregated (Agg-LDL) LDL using a novel magnetic force transducer.Ox-LDL loading reduced the ability of J774A.1 macrophages to generate isometric forces by 50% relative to control cells. Changes in forcefrequency consistent with reduced motility were detected as well.Agg-LDL loading was also detrimental to J774A.1 motility but to alesser extent than Ox-LDL. Ox-LDL loading significantly reduced totalactin levels and induced changes in the F-actin to G-actindistribution, whereas Agg-LDL loaded cells had significantly increasedlevels of total actin. These data provide evidence that cholesterolloading and subsequent accumulation decreases macrophage motility byreducing the cells' force generating capacity and that Ox-LDL appearsto be more effective than Agg-LDL in disrupting the locomotor machinery.

     

Uptake of irradiated human low density lipoprotein by cultured Chinese hamster V79 cells

Specific binding and degradation of native and gamma-rays irradiated (100-2000 rad; 100 rad/min; 137Cs) human low density lipoprotein by Chinese hamster V79 cells and mouse peritoneal macrophage line, J774G were studied. Low density lipoproteins were labeled with 125I for studying the specific binding and subsequent degradation. The specific binding and degradation of irradiated 125I-low density lipoproteins (mixed with irradiated native lipoprotein) by Chinese hamster V79 cells are considerably reduced. The uptake depends on the concentration of thiobarbutaric acid-reactive products generated in the irradiated lipoproteins which in turn depends on the concentration of carotenoids. In contrast the rate of uptake of oxidized low density lipoproteins is enhanced by Chinese hamster macrophages. The alteration in the surface amino groups of apo-B of low density lipoprotein either due to direct damage of peptide bonds by gamma-rays or via interaction with lipid peroxides (generated in the core upon irradiation) are invoked as possible mechanisms for the reduction in specific binding and subsequent degradation by V79 cells.     

Oxidation of chylomicron remnant-like particles inhibits their uptake by THP-1 macrophages by apolipoprotein E-dependent processes

The influence of the oxidative state of chylomicron remnants (CMR) on the mechanisms of their uptake and induction of lipid accumulation by macrophages derived from the human monocyte cell line, THP-1, during foam cell formation was investigated using chylomicron-remnant-like particles (CRLPs) at 3 different levels of oxidation. The oxidative state of CRLPs was varied by exposure to CuSO(4) (oxCRLPs) or incorporation of the antioxidant, probucol (pCRLPs) into the particles. oxCRLPs caused significantly less accumulation of triacylglycerol in the macrophages than CRLPs, and their rate of uptake was lower, while pCRLPs caused more lipid accumulation and were taken up faster. Uptake of all 3 types of particles was inhibited to a similar extent when entry via the low density lipoprotein (LDL) receptor related protein (80-90%), LDL receptor (-30-40%), CD36 (-40%) and phagocytosis (-35-40%) was blocked using lactoferrin, excess LDL, anti-CD36 and cytochalasin D, respectively, but blocking scavenger receptors-A or -B1 using poly inosinic acid or excess HDL had no effect. These findings show that oxidation of CRLPs lowers their rate of uptake and induction of lipid accumulation in macrophages. However, oxidation does not change the main pathways of internalisation of CRLPs into THP-1 macrophages, which occur mainly via the LRP with some contribution from the LDLr, while CD36 and phagocytosis have only a minor role, regardless of the oxidative state of the particles. Thus, the effects of CMR oxidation on foam cell formation contrast sharply with those of LDL oxidation and this may be important in the role of dietary oxidized lipids and antioxidants in modulating atherosclerosis.     

Effects of sphingomyelin on apolipoprotein E- and lipoprotein lipase-mediated cell uptake of lipid particles

It has been reported that human plasma sphingomyelin (SM) levels are positively and independently related to coronary artery disease. The lipoprotein surface is mainly formed by phosphatidylcholine (PC) and SM together with cholesterol and apolipoproteins. However, the influence of SM on the cell uptake of triglyceride-rich lipoproteins and remnants is poorly understood. To clarify the role of SM in lipoprotein uptake, we prepared lipid emulsions containing triolein, PC and SM as model particles of lipoproteins. Apolipoprotein E (ApoE) binding studies revealed that incorporation of SM into the emulsion surface reduced the binding capacity of apoE without changing the affinity. Surface SM reduced apoE-mediated uptake of emulsions by HepG2 cells because of the decreased amount of binding apoE. Apolipoproteins C-II and C-III inhibited the apoE-mediated uptake of SM containing emulsions more effectively. The stimulatory effect of lipoprotein lipase (LPL) on emulsion uptake was decreased by replacing surface PC with SM. These results suggest that SM-induced changes in the binding properties of apolipoproteins and LPL correlate with decreased hepatic uptake of lipid particles.     

Shigella invasion of macrophage requires the insertion of IpaC into the host plasma membrane. Functional analysis of IpaC.

Shigella infects residential macrophages via the M cell entry, after which the pathogen induces macrophage cell death. The bacterial strategy of macrophage infection, however, remains largely speculative. Wild type Shigella flexneri (YSH6000) invaded macrophages more efficiently than the noninvasive mutants, where YSH6000 induced large scale lamellipodial extension including ruffle formation around the bacteria. When macrophages were infected with the noninvasive ipaC mutant, the invasiveness and induction of membrane extension were dramatically reduced as compared with that of YSH6000. J774 macrophages infected with YSH6000 showed tyrosine phosphorylation of several proteins including paxillin and c-Cbl, and this pattern was distinctive from those stimulated by Salmonella typhimurium or phorbol ester. Upon addition of IpaC into the external medium of macrophages, membrane extensions were rapidly induced, and this promoted uptake of Escherichia coli. The exogenously added IpaC was found to be integrated into the host cell membrane as detected by immunostaining. The IpaC domain required for the induction of membrane extension from J774 was narrowed down within the region of residues 117-169, which contains a putative membrane-spanning sequence. Our data indicate that Shigella directs its own entry into macrophages, and the IpaC domain which is required for the association with its host membrane is crucial.     

Relative roles of LDLr and LRP in the metabolism of chylomicron remnants in genetically manipulated mice

Remnant-like emulsions labeled with cholesteryl [(13)C]-oleate were prepared with lipid compositions similar to remnants derived from triacylglycerol-rich lipoproteins. When injected into the bloodstream of conscious mice, the remnant-like emulsions were metabolized in the liver leading to the appearance of (13)CO(2) in the breath. Previously, using this technique, we found that remnant metabolism was significantly impaired but not completely inhibited in mice lacking low density lipoprotein receptors (LDLr). We have now found in mice with non-functional low density lipoprotein receptor-related protein (LRP) that breath enrichment of (13)CO(2) was significantly decreased, indicating that the LRP also plays an important role in the metabolism of chylomicron remnants (CR). The enrichment of (13)CO(2) in the expired breath was negligible in mice lacking both LDLr and receptor-associated protein (-/-), essential for normal function of LRP. In mice pre-injected with gluthatione S-transferase-receptor-associated protein to block LRP binding, there was a marked inhibition of the appearance of (13)CO(2) in the expired breath of homozygous LDLr-deficient mice, supporting the role of LRP in vivo. Whether or not LDLr were present, in mouse and human fibroblast cells human apoE3 or E4 but not apoE2 were essential for binding of remnant-like emulsions, while lactoferrin and suramin completely inhibited binding. We conclude that in normal mice LDLr are important for the physiological metabolism of CR. When LDLr are absent the evidence supports a role for the LRP in the uptake of CR in liver cells and in fibroblasts, with binding characteristics for CR-associated apoE similar to LDLr.     

Macrophage expression of LRP1, a receptor for apoptotic cells and unopsonized erythrocytes, can be regulated by glucocorticoids

Macrophage phagocytosis of apoptotic cells, or unopsonized viable CD47(-/-) red blood cells, can be mediated by the interaction between calreticulin (CRT) on the target cell and LDL receptor-related protein-1 (LRP1/CD91/α2-macroglobulin receptor) on the macrophage. Glucocorticoids (GC) are powerful in treatment of a range of inflammatory conditions, and were shown to enhance macrophage uptake of apoptotic cells. Here we investigated if the ability of GC to promote macrophage uptake of apoptotic cells could in part be mediated by an upregulation of macrophage LRP1 expression. Using both resident peritoneal and bone marrow-derived macrophages, we found that the GC dexamethasone could dose- and time-dependently increase macrophage LRP1 expression. The GC receptor-inhibitor RU486 could dose-dependently prevent LRP1 upregulation. Dexamethasone-treated macrophages did also show enhanced phagocytosis of apoptotic thymocytes as well as unopsonized viable CD47(-/-) red blood cells, which was sensitive to inhibition by the LRP1-agonist RAP. In conclusion, these data suggest that GC-stimulated macrophage uptake of apoptotic cells may involve an upregulation of macrophage LRP1 expression and enhanced LRP1-mediated phagocytosis.     

Constitutive receptor-independent low density lipoprotein uptake and cholesterol accumulation by macrophages differentiated from human monocytes with macrophage-colony-stimulating factor (M-CSF)

Recently, we have shown that macrophage uptake of low density lipoprotein (LDL) and cholesterol accumulation can occur by nonreceptor mediated fluid-phase macropinocytosis when macrophages are differentiated from human monocytes in human serum and the macrophages are activated by stimulation of protein kinase C (Kruth, H. S., Jones, N. L., Huang, W., Zhao, B., Ishii, I., Chang, J., Combs, C. A., Malide, D., and Zhang, W. Y. (2005) J. Biol. Chem. 280, 2352-2360). Differentiation of human monocytes in human serum produces a distinct macrophage phenotype. In this study, we examined the effect on LDL uptake of an alternative macrophage differentiation phenotype. Differentiation of macrophages from human monocytes in fetal bovine serum with macrophage-colony-stimulating factor (M-CSF) produced a macrophage phenotype demonstrating constitutive fluid-phase uptake of native LDL leading to macrophage cholesterol accumulation. Fluid-phase endocytosis of LDL by M-CSF human macrophages showed non-saturable uptake of LDL that did not down-regulate over 48 h. LDL uptake was mediated by continuous actin-dependent macropinocytosis of LDL by these M-CSF-differentiated macrophages. M-CSF is a cytokine present within atherosclerotic lesions. Thus, macropinocytosis of LDL by macrophages differentiated from monocytes under the influence of M-CSF is a plausible mechanism to account for macrophage foam cell formation in atherosclerotic lesions. This mechanism of macrophage foam cell formation does not depend on LDL modification or macrophage receptors.