Adhesion of monocytes to LRP clusters II and IV. Freshly isolated monocytes were stimulated with 100 nM PMA for 15 minutes and added to immobilized LRP cluster II or IV for 30 minutes at room temperature in the presence or absence of LRP-antagonist GST-RAP (50 μg/mL). (A) Typical experiment visualized by light microscopy, in which 1.5 × 105 cells were added to each well. Original magnification, 400×. (Bi) To quantify cell adhesion, different amounts of cells (0-1.5 × 105 cells/well) were added. After incubation and subsequent washing, bound cells were lysed using 1% Triton-X100/50 mM acetic acid (pH 5.0), and endogenous alkalic phosphatase activity was determined using PNP as substrate. ○ indicates LRP cluster II; •, LRP cluster IV. (Bii) Relative adhesion in the presence (▪) or absence (□) of GST-RAP. Data are corrected for adhesion to uncoated wells (less than 20% of cluster IV coated wells) and represent mean ± SD of 3 experiments performed in duplicate. (C) PMA-stimulated U937 cells (1.5 × 106) were incubated with indicated antibodies (20 μg/mL), fibrinogen (50 μg/mL), or vitronectin (100 μg/mL) for 15 minutes, or wells were preincubated with GST-RAP (50 μg/mL) and added to immobilized IV for 60 minutes at 37°C. Adhered cells were detected as described in panel B. Presented is the percentage of adhesion relative to adhesion in the absence of antibodies or GST-RAP. Data represent the mean ± SD of 3 to 10 experiments performed in duplicate. ns indicates not significant (P > .05).
