bioRxiv2025
The pentameric C-reactive protein (pCRP), an acute-phase protein, binds to lysophosphatidylcholine (LPC) displayed on the surface of dying cells and microorganisms to activate the complement system and to opsonize immune cells via Fc{gamma}-receptors (Fc{gamma}Rs). Members of the Fc{gamma}R family are characterized by the recognition of the Fc part of IgG antibodies. We utilized a mouse thymoma BW5147 reporter cell panel stably expressing chimeric human Fc{gamma}R-CD3{zeta}-chain receptors to define the molecular requirements for Fc{gamma}R crosslinking by C-reactive protein (CRP). Applying this approach, we show a robust activation of CD64/Fc{gamma}RI and CD32a/Fc{gamma}RIIa by immobilized CRP isoforms as well as triggering of inhibitory CD32b/Fc{gamma}RIIb. Of note, activation of Fc{gamma}RIIa was restricted to the 131R allelic variant but not observed with 131H. In contrast, Fc{gamma}RIII isoforms CD16aF, CD16aV and CD16b were not activated by pCRP, although binding of CRP isoforms to Fc{gamma}RIII was detectable. Activation of Fc{gamma}Rs by free pCRP in solution phase was considerably lower than with immobilized pCRP on hydrophilic plastic surfaces and readily abolished by IgG at serum level concentrations, whereas it was enhanced by the addition of streptococci. The types of Fc{gamma}Rs mainly responding to pCRP in solution phase (CD64/Fc{gamma}RI and CD32aR/Fc{gamma}RIIaR) clearly differed from Fc{gamma}Rs responding to soluble multimeric IgG complexes (i.e., CD16aV/Fc{gamma}RIIIaV and CD32aH/Fc{gamma}RIIaH). Compared to pCRP, monomeric CRP (mCRP) showed lower levels of activation in those selective Fc{gamma}Rs. Fc{gamma}R activation was linked to recognition by conformation-dependent CRP antibodies. Unmasking of the mAb 9C9-defined neoepitope in pCRP* correlated with the triggering of Fc{gamma}Rs, indicating that pCRP* is the major Fc{gamma}R-activating CRP conformation. The assay provides a novel, scalable approach to determine the molecular properties of CRP as a physiological ligand of Fc{gamma}R-mediated bioactivities. Scope statementFc{gamma} receptors (Fc{gamma}Rs) are important immune regulators that come in different variants and combinations, making it difficult to predict which components will ultimately lead to immunological effector functions. Classical Fc{gamma}Rs are defined by their recognition of IgG-Fc, while other ligands, such as C-reactive protein (CRP), are often neglected. Circulating concentrations of CRP, an acute phase protein, are elevated during inflammatory responses. As a pattern recognition receptor, CRP binds to lysophosphatidylcholine expressed on the surface of dying cells and microbes in order to activate the complement system via C1q. We have established a reporter cell assay platform that goes beyond ligand binding and takes a deeper look at the activation outcome(s) by CRP compared with IgG-Fc. This is the first comprehensive study defining CRP-responsive vs non-responsive Fc{gamma}Rs and investigating the interaction of Fc{gamma}Rs with CRP isoforms (pCRP/pCRP*/mCRP). We distinguish binding from receptor triggering using reporter cells stably expressing a chimeric Fc{gamma}R-CD3{zeta} chain, thereby defining the molecular requirements for Fc{gamma}R cross-linking by CRP. The assay provides a novel, sensitive and scalable approach to the properties of CRP as a ligand inducing Fc{gamma}R-mediated bioactivities.