Figure 1
Complex formation between CRP and LPC and the impact of the CRP-LPC complex on FcγRs binding kinetics and macrophage activities. (A). CRP bound to LPC-coated plate was detected with a specific anti-CRP antibody and alkaline phosphatase-labelled secondary antibody. Data were expressed as relative light units over 100 ms (RLU/100 ms). The figure represents one of three independent experiments. (B). LPC in the presence or absence of CRP (10 μg/ml each in the presence of 2 mM calcium) was detected by HPLC using an Apollo Silica 5u column. Data are expressed as absorbance units (AU) at 205 nm over time. The figure represents one of three independent experiments. (C and D). The specific binding of CRP or CRP-LPC (up to 10 μg/ml) to 293FT cells transfected with FcγRIA (C) or FcγRIIA (D) was obtained as described in the Methods section. Data shown are mean values ± S.D. from three independent experiments. Kd and Bmax values were analysed using Prism software. (E). H2DCFDA-labelled human macrophages were stimulated with 10 μg/ml CRP, LPC or CRP-LPC complexes for 30 min and the generated intracellular ROS (red > yellow > green > white in the upper panel) was detected by confocal microscopy. Data represent mean values ± S.D. of the amount of intracellular ROS from three independent experiments. (F). NF-kB and AP-1 activity of human macrophages treated with 10 μg/mL CRP, LPC, or CRP-LPC complexes were measured by EMSA. The EMSA image represents one of three independent experiments. Data represent mean values ± S.D. from three independent experiments.