Human Fcγ-receptors selectively respond to C-reactive protein (CRP) isoforms
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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 Fcγ-receptors (FcγRs). Members of the FcγR 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 FcγR-CD3ζ-chain receptors to define the molecular requirements for FcγR crosslinking by C-reactive protein (CRP). Applying this approach, we show a robust activation of CD64/FcγRI and CD32a/FcγRIIa by immobilized CRP isoforms as well as triggering of inhibitory CD32b/FcγRIIb. Of note, activation of FcγRIIa was restricted to the 131R allelic variant but not observed with 131H. In contrast, FcγRIII isoforms CD16aF, CD16aV and CD16b were not activated by pCRP, although binding of CRP isoforms to FcγRIII was detectable. Activation of FcγRs 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 FcγRs mainly responding to pCRP in solution phase (CD64/FcγRI and CD32aR/FcγRIIaR) clearly differed from FcγRs responding to soluble multimeric IgG complexes (i.e., CD16aV/FcγRIIIaV and CD32aH/FcγRIIaH). Compared to pCRP, monomeric CRP (mCRP) showed lower levels of activation in those selective FcγRs. FcγR activation was linked to recognition by conformation-dependent CRP antibodies. Unmasking of the mAb 9C9-defined neoepitope in pCRP* correlated with the triggering of FcγRs, indicating that pCRP* is the major FcγR-activating CRP conformation. The assay provides a novel, scalable approach to determine the molecular properties of CRP as a physiological ligand of FcγR-mediated bioactivities.
Scope statement
Fcγ receptors (FcγRs) 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 FcγRs 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 FcγRs and investigating the interaction of FcγRs with CRP isoforms (pCRP/pCRP*/mCRP). We distinguish binding from receptor triggering using reporter cells stably expressing a chimeric FcγR-CD3ζ chain, thereby defining the molecular requirements for FcγR cross-linking by CRP. The assay provides a novel, sensitive and scalable approach to the properties of CRP as a ligand inducing FcγR-mediated bioactivities.
