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. 2010 Oct;30(10):2022-31.
doi: 10.1161/ATVBAHA.110.210849. Epub 2010 Jul 15.

Native low-density lipoprotein uptake by macrophage colony-stimulating factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis

Joshua J Anzinger  1 , Janet Chang, Qing Xu, Chiara Buono, Yifu Li, Francisco J Leyva, Bum-Chan Park, Lois E Greene, Howard S Kruth
Affiliations

Native low-density lipoprotein uptake by macrophage colony-stimulating factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis

Joshua J Anzinger et al. Arterioscler Thromb Vasc Biol. 2010 Oct.

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.

Conclusions: 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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Figures

Figure 1
Figure 1
A through D, SU6656 or toxin B only partially inhibits macrophage LDL uptake and cholesterol accumulation. Macrophages were incubated for 24 hours with 250 µg/mL 125I-LDL and the indicated addition (A and C) or 1 mg/mL LDL and the indicated addition (B and D). 125I-LDL uptake is the sum of cell-associated and degraded 125I-LDL. To determine basal macrophage cholesterol levels, macrophages were also incubated for 24 hours without LDL addition. *P< 0.05 for LDL vs LDL plus added inhibitor.
Figure 2
Figure 2
A through D, SU6656 or toxin B almost completely inhibits macrophage macropinosome formation. Macrophages were treated for 5 hours without SU6656 (A) or with 20 µmol/L SU6656 (B) or for 5 hours without toxin B (C) or with 100 ng/mL toxin B (D) and imaged by phase-contrast microscopy. The macropinosome vacuoles were substantially decreased in cultures with added inhibitor. The bar indicates 50 µm.
Figure 3
Figure 3
LDL uptake by macrophages is efficiently inhibited by a combination of macropinocytosis and micropinocytosis inhibitors. Macrophages were incubated for 5 hours in medium containing 250 µg/mL 125I-LDL and the indicated inhibitor. The percentage inhibition of uptake compared with control is shown. 125I-LDL uptake for untreated macrophages was 10 µg/mg cell protein.
Figure 4
Figure 4
A through D, Macrophages take up the fluid-phase pinocytosis tracer HRP within both macropinosomes and micropinosomes. Macrophages were incubated for 10 minutes without HRP (A) or with 1 mg/mL HRP (B) without inhibitor addition, with 10 µmol/L SU6656 (C), or with 500 nmol/L bafilomycin A1 (D). SU6656 inhibited the formation of macropinosomes but not micropinosomes, whereas bafilomycin A1 inhibited the formation of micropinosomes but not macropinosomes. The bar indicates 10 µm.
Figure 5
Figure 5
A through D, Electron microscopy of macrophage uptake of the fluid-phase pinocytosis tracer HRP within macropinosomes and micropinosomes. Macrophages were incubated for 10 minutes without HRP as a control (A) or with 1 mg/mL HRP (B) without inhibitor addition, with 10 µmol/L SU6656 (C), or with 500 nmol/L bafilomycin A1 (D). Arrows and arrowheads indicate macropinosomes and micropinosomes, respectively. The bar indicates 0.5 µm.
Figure 6
Figure 6
A through D, Macrophages take up LDL within macropinosomes and micropinosomes. Macrophages were incubated for 10 minutes with 5 mg/mL LDL. LDL detected with anti-LDL immunogold labeling was observed in both macropinosomes (A) and micropinosomes (B and C). As a control, samples were treated with anti–green fluorescent protein immunogold labeling and this showed no labeling (D). The bar indicates 0.2 µm.
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