A phagocytosis assay for oxidized low-density lipoprotein versus immunoglobulin G-coated microbeads in human U937...

The human monocyte cell line U937 was differentiated into an adherent macrophage phenotype using phorbol 12-myristate 13-acetate (PMA) to assay the phagocytosis of oxidized low-density lipoprotein (oxLDL) that may play a role in atherosclerosis. Microbeads were coated with the inflammatory ligand oxLDL to create a novel phagocytosis assay that models the binding of macrophages to oxLDL in the solid phase such as found in the fatty streaks of the arteries. The oxLDL was prepared with LDL from human ethylenediaminetetraacetic acid (EDTA) plasma oxidized with an excess (5 mM) of the strong oxidizing agent CuSO4 and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Western blot. The binding of the oxLDL to the beads was confirmed by DilC18-oxLDL staining and confocal microscopy in addition to trypsin digestion of the microbeads for liquid chromatography, electrospray ionization, and tandem mass spectrometry. Phagocytosis of the oxLDL versus human bulk immunoglobulin G1 (IgG1)-coated microbeads was assayed over time, in the presence and absence of serum factors, by pulse chase and with enzyme inhibitor treatments. The ligand beads were then stained with specific antibodies to oxLDL versus human IgG to differentially stain external versus engulfed ligand microbeads. The phagocytosis of oxLDL and IgG ligand microbeads was abolished by the actin polymerization inhibitors cytochalasin D and latrunculin. Pharmacological inhibitors of the receptor enzymes JAK, SRC, and PLC prevented both IgG and oxLDL receptor function. In contrast, the function of the oxLDL phagocytic receptor complex was more sensitive to inhibition of PTK2, PKC, and SYK activity.