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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:2372.)
© 2004 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Ultrasensitive Confocal Fluorescence Microscopy of C-Reactive Protein Interacting With FcRIIa

Dimitar E. Manolov; Carlheinz Röcker; Vinzenz Hombach; G. Ulrich Nienhaus; Jan Torzewski

From the Departments of Internal Medicine II–Cardiology (D.E.M., V.H., J.T.) and Biophysics (C.R., G.U.N.), University of Ulm, Germany; and the Department of Physics (G.U.N.), University of Illinois at Urbana-Champaign, Urbana, Ill.

Correspondence to G. Ulrich Nienhaus, Professor, PhD, University of Ulm, Department of Biophysics, 89069 Ulm, Germany. E-mail uli{at}uiuc.edu

Background— C-Reactive protein (CRP) is an acute phase protein with a suggested pathogenic role in cardiovascular disease. Previous reports proposed that the low-affinity IgG receptor FcRIIa is the major receptor for CRP. However, these reports were met with criticism because the use of anti-CRP antibodies in the detection of CRP binding to FcRIIa may have caused false-positive results.

Methods and Results— To resolve this controversy, we used ultrasensitive fluorescence microscopy to study the association, dissociation, and equilibrium of CRP binding to FcRIIa. CRP indeed binds to FcRIIa, with low association rates and dissociation rates. Anti-CRP antibodies markedly enhance binding, as is evident from the decrease of the equilibrium dissociation coefficient by 2 orders of magnitude.

Conclusions— Our study demonstrates the virtues of single fluorophore labeling and highlights the pitfalls of immunolabeling in investigating CRP/Fc receptor interactions. Importantly, this article provides the first quantitative characterization of CRP binding to FcRIIa and explains and reconciles the diverse and conflicting data presented in the literature.

We have studied CRP binding to FcRIIa using the novel method of ultrasensitive confocal fluorescence microscopy. We unambiguously show that CRP interacts with FcRIIa and characterize this interaction quantitatively. We also provide explanations as to why controversial results were obtained previously.

Key Words: atherosclerosis • inflammation • receptors • ultrasensitive fluorescence microscopy

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