Binding and internalization of C-reactive protein by Fcgamma receptors on human aortic endothelial cells mediates biological effects.
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).


RESULTS
Binding studies were performed by incubation of HAECs with biotinylated CRP (B-CRP, 25 to 200 microg/mL) for 30 to 180 minutes at 4 degrees 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 microg/mL and Kd of 88 nM. CRP binding was inhibited by 10x cold CRP (58%). CRP (100 microg/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.
Devaraj
Sridevi
S
Laboratory for Atherosclerosis and Metabolic Research, Department of Pathology, UC Davis Medical Center, Sacramento, Calif 95817, USA.
Du Clos
Terry W
TW
Jialal
Ishwarlal
I
D000906:Antibodies / Q000494:pharmacology; D001011:Aorta / Q000166:cytology; D050197:Atherosclerosis / Q000276:immunology*; D019921:Biotinylation; D002097:C-Reactive Protein / Q000378:metabolism*; D002478:Cells, Cultured; D004705:Endocytosis / Q000276:immunology; D004730:Endothelium, Vascular / Q000166:cytology / Q000276:immunology* / Q000378:metabolism*; D011464:Epoprostenol / Q000378:metabolism; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D007962:Leukocytes / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D017452:Receptors, IgG / Q000276:immunology / Q000378:metabolism*; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
D000906:Antibodies; D016209:Interleukin-8; D017452:Receptors, IgG; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1; D002097:C-Reactive Protein; D011464:Epoprostenol; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III