Complement-mediated phagocytosis using macrophage-like differentiated HL60 cells and serum-treated zymosan. (A) Expression of the CR3/integrin αM in macrophage-like differentiated HL60 cells. HL60 cells were treated with 10–7 M vitamin D3 (VD3) and 10 ng/mL TPA for indicated days and the expression of the CR3/integrin αM and Syk was examined by immunoblotting (IB) analysis with the corresponding antibodies. The blot is a representative of 3 independent experiments. (B) Binding of C3bi to zymosan. To opsonize zymosan with C3bi, zymosan was incubated in 50% human serum at 37°C for 30 minutes in the presence or absence of compstatin or control peptide and then washed with PBS twice at 4°C. For comparison, zymosan was also treated with PBS at 37°C for 30 minutes or in 50% human serum at 4°C. Binding of C3bi to zymosan was confirmed by flow cytometry with anti-C3bi antibody (thick line) or with control mouse IgG (thin line). The representative flow cytometric patterns (left) and the ratio of mean fluorescence intensity (anti-C3bi antibody/control IgG) with SD of triplicate experiments at the indicated conditions (right) are presented. The statistically significant difference was assessed by the Student t test; *P < .05. (C) Phagocytosis of zymosan particles by HL60 cells. Serum-opsonized (top panel) or nonopsonized zymosan (bottom panel) was added to macrophage-like differentiated Day3-HL60 cells (cell-zymosan ratio, 1:10) and incubated at 37°C. Phagocytosis was recorded by a time-lapse microscope every 15 seconds and analyzed by MacSCOPE image analyzing software. An LCPlan 20 ×/0.40 numeric aperture (NA) objective was used to visualize the images. The bar indicates 10 μm.
