Native Low-Density Lipoprotein Uptake by Macrophage Colony-Stimulating Factor–Differentiated Human Macrophages Is Mediated by Macropinocytosis and Micropinocytosis

Abstract

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.

Murine bone marrow-derived macrophages differentiated with GM-CSF become foam cells by PI3Kγ-dependent fluid-phase pinocytosis of native LDL

“…Although one or more of these components may mediate PI3K ␥ -independent macropinocytosis for murine GM-CSF-differentiated macrophages, these components may mediate macropinocytosis only in certain cell types. For example, previous studies show a role for Src family kinases in mediating macropinocytosis in several cell lines ( 38,39 ), whereas we previously observed that human M-CSF-differentiated macrophages show Src family kinase-independent macropinocytosis ( 10 ). Future studies are warranted to identify the PI3K ␥ -independent pathways mediating macropinocytosis for murine GM-CSF-differentiated macrophages.…”

“…After three washes with DPBS, macrophages were mounted with Vectashield containing DAPI (catalog number H-1500, Vector) and examined by fl uorescence microscopy. For identifi cation of CD68, macrophages were incubated with 10% goat serum in DPBS for 30 min, then rinsed macropinocytosis and micropinocytosis to become foam cells ( 10 ). Macrophages differentiated with human serum have a granulocyte-macrophage colony-stimulating factor (GM-CSF)-differentiated phenotype, and when activated with PMA, primarily take up LDL by macropinocytosis to become foam cells ( 6,11 ).…”

Low-density Lipoprotein Receptor Deficiency Causes Impaired Osteoclastogenesis and Increased Bone Mass in Mice because of Defect in Osteoclastic Cell-Cell Fusion

“…Numerous studies have indicated a critical involvement of cholesterol in the differentiation and function of a variety of cell types, including endothelial cells, cardiac muscle cells, macrophages, and osteoblasts (57)(58)(59). It is well known that cholesterol is a major component of the plasma membrane, and especially of lipid rafts (36 -39).…”

Dynamic T cell–APC interactions sustain chronic inflammation in atherosclerosis

“…Disruption of T cell-APC interaction by the addition of an MHCII blocking antibody decreased oxLDL accumulation in CD11b + CD11c -cells (Figure 7, C and D). mmLDL is also taken up by macrophages, but uses a different mechanism (62,63). mmLDL uptake by macrophages was enhanced in the presence of model antigen ( Figure 7, E and F), and this was reduced in the presence of a blocking MHCII antibody ( Figure 7, G and H).…”