Binding and Internalization of C-Reactive Protein by Fcgamma Receptors on Human Aortic Endothelial Cells Mediates Biological Effects

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Abstract
Objective— In addition to being a cardiovascular risk marker, recent studies support a role for CRP in atherothrombosis. Several investigators have reported that CRP binds to Fcgamma receptors on leukocytes. The aim of the study is to determine the processing of CRP by human aortic endothelial cells (HAECs). Methods and Results— Binding studies were performed by incubation of HAECs with biotinylated CRP (B-CRP, 25 to 200 μg/mL) for 30 to 180 minutes at 4°C. B-CRP binding was quantitated using streptavidin-fluorescein isothiocyanate followed by flow cytometry. Saturable binding of CRP was obtained at 60 minutes with a CRP concentration between 100 to 150 μg/mL and Kd of 88 nM. CRP binding was inhibited by 10× cold CRP (58%). CRP (100 μg/mL) significantly upregulated surface expression of Fcgamma receptors, CD32, as well as CD64 on HAECs (PConclusions— We demonstrate that CRP mediates its biological effects on HAECs via binding and internalization through Fcgamma receptors, CD32 and CD64. In addition to being a risk marker, CRP exerts atherothrombotic effects in endothelial cells. In this study, using flow cytometry and fluorescence microscopy, we show that CRP binds to CD32 and CD64 on HAECs, is internalized, and exerts its biological effects. Antibodies to CD32 and CD64 abrogated the biological effects of CRP, whereas antibodies to CD16 had no effect.