Macrophage cholesterol accumulation is considered a critical process in the development of atherosclerotic plaques, the cause of most heart attacks and strokes. LDL, 2 the main carrier of blood cholesterol, enters the blood vessel wall and by some mechanism, monocyte-derived macrophages take up the LDL in the vessel wall. Macrophage cholesterol accumulation converts the macrophages into so-called foam cells and stimulates the macrophages to secrete proteases and tissue factor that contribute to plaque rupture and thrombosis, respectively (1-3).Because incubation of macrophages with native LDL did not produce substantial macrophage cholesterol accumulation in past studies, it was proposed that LDL modifications that promote recognition and uptake of large amounts of the modified LDL by macrophage scavenger receptors is required to generate macrophage foam cells (4). LDL oxidation has been the most commonly studied LDL modification that stimulates LDL uptake (5).Recently, we have shown that treatment of human monocyte-derived macrophages with protein kinase C activators such as PMA stimulates fluid-phase macropinocytosis of native (unmodified) LDL (6, 7). This results in uptake of LDL in amounts directly proportional to its concentration in the medium. As a result, incubation of human monocytederived macrophages with LDL at concentrations similar to those that exist within the normal vessel wall (0.7-2.7 mg/ml) (8 -10) leads to macrophage foam cell formation.Human monocytes can differentiate into macrophage phenotypes with different morphology and function depending on the culture conditions and differentiation factors included in the culture medium (11). In the studies discussed above, we differentiated human monocytes into macrophages in the presence of human serum, producing the macrophage phenotype that resembles a "fried egg." In the present study, we show that differentiation of human monocytes into macrophages in the presence of fetal bovine serum with added M-CSF produces a macrophage phenotype having an elongated shape and showing high levels of constitutive macropinocytosis not dependent on activation of the macrophage with PMA. The constitutive macropinocytosis in these macrophages mediated fluid-phase uptake of LDL causing high levels of macrophage cholesterol accumulation. Thus, our findings demonstrate a macrophage model system showing constitutive cholesterol accumulation without the need for either LDL modification or LDL uptake mediated by receptors. MATERIALS AND METHODSCulture of Human Monocyte-derived Macrophages-Human monocytes were purified with counterflow centrifugal elutriation of mononuclear cells obtained by monocytopheresis of normal human donors. M-CSF monocyte-derived macrophage cultures were begun with the elutriated human monocytes suspended in RPMI 1640 medium (MediaTech) with 10% fetal bovine serum (FBS) (Invitrogen) seeded in 6-well plates (MatTek and Corning) at a density of 4 ϫ 10 5 monocytes/cm 2 . After 2-h incubation in a cell culture incubator with 5% CO 2 /95% air at 37°C,...

Objective To examine the pinocytotic pathways mediating native low-density lipoprotein (LDL) uptake by human macrophage colony-stimulating factor–differentiated macrophages (the predominant macrophage phenotype in human atherosclerotic plaques). Methods and Results We identified the kinase inhibitor SU6656 and the Rho GTPase inhibitor toxin B as inhibitors of macrophage fluid-phase pinocytosis of LDL. Assessment of macropinocytosis by time-lapse microscopy revealed that both drugs almost completely inhibited macropinocytosis, although LDL uptake and cholesterol accumulation by macrophages were only partially inhibited (approximately 40%) by these agents. Therefore, we investigated the role of micropinocytosis in mediating LDL uptake in macrophages and identified bafilomycin A1 as an additional partial inhibitor (approximately 40%) of macrophage LDL uptake that targeted micropinocytosis. When macrophages were incubated with both bafilomycin A1 and SU6656, inhibition of LDL uptake was additive (reaching 80%), showing that these inhibitors target different pathways. Microscopic analysis of fluid-phase uptake pathways in these macrophages confirmed that LDL uptake occurs through both macropinocytosis and micropinocytosis. Conclusion Our findings show that human macrophage colony-stimulating factor–differentiated macrophages take up native LDL by macropinocytosis and micropinocytosis, underscoring the importance of both pathways in mediating LDL uptake by these cells.

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