Nancy L. Jones

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.

The Pathogenesis of Foam Cell Formation Modified LDL Stimulates Uptake of Co-Incubated LDL Via Macropinocytosis

Previously, modified LDLs were shown to stimulate macropinocytosis in pigeon macrophages. Simultaneous intracellular trafficking of LDL and AcLDL, differentially labeled with colloidal gold, was done to determine whether uptake of LDL, which does not cause foam cell formation, was internalized via a separate route from AcLDL, which stimulates foam cell formation. AcLDL and LDL were followed at either low (12 microg/mL) concentrations near the saturation of high affinity binding sites or high (50 to 150 microg/mL) lipoprotein concentrations used to induce foam cell formation. The colloidal gold distribution and percentage of co-labeling as observed by transmission electron microscopy were determined for organelles involved with coated-pit endocytosis or macropinocytosis. LDL simultaneously incubated with AcLDL on macrophages at the low concentration was predominately internalized via coated-pit endocytosis. AcLDL was internalized via both coated-pit endocytosis and macropinocytosis at low concentration. At higher lipoprotein concentrations (50 to 150 microg/mL), AcLDL continued to be internalized via macropinocytosis. Interestingly, a significant portion of the co-incubated LDL, at high concentrations, also trafficked via macropinocytosis. LDL internalized by macropinosomes at high lipoprotein concentrations suggests that AcLDL-stimulated macropinocytosis might increase uptake of co-incubated lipoproteins. When (125)I-LDL was incubated with cold AcLDL, LDL degradation at 37 degrees C doubled, without a corresponding increase in cell association or total binding of LDL at 4 degrees C. These studies suggest that modified LDL-stimulated macropinocytosis is a mechanism for increased degradation of co-incubated LDL potentially leading to foam cell formation.

Modified LDLs are internalized by macrophages in part via macropinocytosis

Macrophage foam cell formation in vitro requires uptake of modified low density lipoproteins (LDL) such as acetylated LDL (AcLDL) and moderately oxidized LDL (OxLDL). Macrophages incubated with AcLDL and OxLDL, but not LDL, showed increased membrane ruffling as seen with time‐lapse phase contrast video light microscopy. Modified LDLs stimulated circular membrane ruffles between 2 and 10 min after incubation. These membrane ruffles were readsorbed into the plasma membrane between 5 and 15 min later. Phase‐bright macropinosomes formed at the base of the stimulated membrane ruffles. The fluid‐phase marker lucifer yellow labeled the modified LDL stimulated macropinosomes. Modified LDLs stimulate fluid‐phase uptake by 1.5‐fold to threefold as measured with 14C‐sucrose uptake. Transmission electron microscopy showed that gold conjugated AcLDL and OxLDL bound preferentially to membrane ruffles and were present in macropinosomes (diameter >0.2 μm) underneath these membrane ruffles. AcLDL and OxLDL were also present in clathrin‐coated pits and endosomes. These studies suggest that modified lipoproteins stimulate macropinocytosis. AcLDL and OxLDL are partially internalized by macropinocytosis and partially internalized via clathrin‐coated pit endocytosis. Anat Rec 255:57–68, 1999.

Modified LDLs Induce and Bind to Membrane Ruffles on Macrophages

Macrophage foam cell formation in vitro requires uptake of modified low density lipoproteins (LDL) such as acetylated LDL (AcLDL) and moderately oxidized LDL (OxLDL), or beta‐migrating very low density lipoprotein (βVLDL), a naturally occurring lipoprotein. Incubation of macrophages with AcLDL and OxLDL resulted in stimulation of membrane ruffle formation, while βVLDL primarily resulted in increased numbers of microvilli. Time‐lapse Allen video enhanced contrast differential interference contrast (AVEC‐DIC) light microscopy and correlative whole mount intermediate‐voltage transmission electron microscopy (IVEM) was used to examine the dynamics of AcLDL stimulated membrane ruffling and membrane ruffle ultrastructure. Stereo 3D surface replicas confirmed that AcLDL bound to these AcLDL‐induced membrane ruffles. Quantification of the plasma membrane surface area after incubation with AcLDL, βVLDL or LDL confirmed that AcLDL stimulated membrane ruffling, while βVLDL and LDL stimulated microvilli formation. These studies suggest that modified LDLs induce circular membrane ruffles and modified LDLs bind to these ligand‐induced membrane ruffles. Anat Rec 255:44–56, 1999.

The LDL receptor and LRP are receptors for βVLDL on pigeon monocyte-derived macrophages

Receptors for the lipoprotein, beta very low density lipoprotein (βVLDL), have been identified through the binding of βVLDL-gold conjugates on two ligand-induced regions of pigeon monocyte-derived macrophages. These regions were microvilli/retraction fibers and membrane ruffles. The present study investigated the location and identity of βVLDL receptors using an antiserum directed against the epidermal growth factor (EGF) precursor region of the human low density lipoprotein (LDL) receptor. The anti-receptor serum recognized two membrane proteins from pigeon monocyte-derived macrophages, a 116 kDa (LDL receptor) protein and a 600 kDa (low density lipoprotein receptor-related protein; LRP) protein. Ligand blot analysis demonstrated that pigeon βVLDL bound to both the LDL receptor and LRP. Immuno-gold electron microscopy using the anti-receptor serum resulted in immunoglobulin localization on the same two ligand-induced regions, microvilli/retraction fibers and membrane ruffles, to which the ligand had bound. Furthermore, simultaneous immunogold localization of the lipoprotein receptor antigens and βVLDL-gold (ligand) binding substantiated co-localization of the receptor antigens and βVLDL on the ligand-induced regions. Cross-competition studies with the anti-receptor serum and βVLDL-gold conjugates documented that increasing concentration of the anti-receptor serum resulted in 70% inhibition of βVLDL-gold conjugate binding. These data suggest that pigeon monocyte-derived macrophages utilize both the LDL receptor and LRP as receptors for pigeon βVLDL.