Published online before print April 28, 2005, doi: 10.1161/01.ATV.0000168573.10844.ae
© 2005 American Heart Association, Inc.
Vascular Biology |
Binding and Internalization of C-Reactive Protein by Fcgamma Receptors on Human Aortic Endothelial Cells Mediates Biological Effects
From the Laboratory for Atherosclerosis and Metabolic Research, Department of Pathology (S.D., I.J.), UC Davis Medical Center, Sacramento, Calif; and the Department of Medicine (T.W.D.C.), University of New Mexico and Veterans Affairs Medical Center, Albuquerque, N.M.
Correspondence to Ishwarlal Jialal, MD, PhD, Director of the Laboratory for Atherosclerosis and Metabolic Research, 4635 II Avenue, Res 1 Bldg, Rm 3000, Sacramento, CA 95817. E-mail ishwarlal.jialal{at}ucdmc.ucdavis.edu
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 10x cold CRP (58%). CRP (100 µg/mL) significantly upregulated surface expression of Fcgamma receptors, CD32, as well as CD64 on HAECs (P<0.01). Also, preincubation with anti-CD32 and CD64 antibodies significantly inhibited maximal binding of CRP to HAECs 64% and 30%, respectively, whereas antibodies to CD16 had no effect. Internalization of CRP, as determined by loss of surface expression, was 50%. Also, binding and internalization of biotinylated CRP was confirmed by confocal microscopy and CRP colocalized with CD32 and CD64. Most importantly, the increase in interleukin-8, intercellular adhesion molecule 1, and vascular cell adhesion molecule-1 and the decrease in eNOS and prostacyclin induced by CRP was abrogated with antibodies to CD32 and CD64.
Conclusions— 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.
Key Words: inflammation • C-reactive protein • Fcgamma receptors • endothelial cells • atherothrombosis
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