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The complement system is a major component of our innate immune system, in which the complement proteins C5a and C5a-des Arg bind to two G-protein-coupled receptors: namely, the C5a receptor (C5a1) and C5a receptor like-2 receptor (C5a2, formerly called C5L2). Recently, it has been demonstrated that C5a, but not C5a-des Arg, upregulates heteromer formation between C5a1 and C5a2, leading to an increase in IL-10 release from human monocyte-derived macrophages (HMDMs). A bioluminescence resonance energy transfer (BRET) assay was used to assess the recruitment of β-arrestins by C5a and C5a-des Arg at the C5a1 and C5a2 receptors. C5a demonstrated elevated β-arrestin 2 recruitment levels in comparison with C5a-des Arg, whereas no significant difference was observed at C5a2. A constitutive complex that formed between β-arrestin 2 and C5a2 accounted for half of the BRET signal observed. Interestingly, both C5a and C5a-des Arg exhibited higher potency for β-arrestin 2 recruitment via C5a2, indicating preference for C5a2 over C5a1. When C5a was tested in a functional ERK1/2 assay in HMDMs, inhibition of ERK1/2 was observed only at concentrations at or above the EC50 for heteromer formation. This suggested that increased recruitment of the β-arrestin-C5a2 complex at these C5a concentrations might have an inhibitory role on C5a1 signaling through ERK1/2. An improved understanding of C5a2 modulation of signaling in acute inflammation could be of benefit in the development of ligands for conditions such as sepsis.

Receptors for C5a have an important role in innate immunity and inflammation where their expression and activation is tightly regulated. There are two known receptors for C5a: the C5a receptor (C5aR) and the C5a receptor like-2 (C5L2) receptor. Here we hypothesized that activation of C5aR might lead to heteromer formation with C5L2, as a downregulatory mechanism for C5aR signaling. To investigate this experimentally, bioluminescent resonance energy transfer (BRET) was implemented and supported by wide-field microscopy to analyze receptor localization in transfected HEK293 cells and human monocyte-derived macrophages (HMDM). BRET experiments indicated the presence of constitutive C5aR-C5L2 heteromers, where C5a, but not C5a-des Arg, was able to induce further heteromer formation, which was inhibited by a C5aR-specific antagonist. The data obtained suggest that C5aR-C5L2 can form heteromers in a process enhanced by C5a, but not by C5a-des Arg. There was also a significant difference in the levels of the anti-inflammatory cytokine IL-10 detected in HMDM following exposure to C5a compared with that seen for C5a-des Arg but no differences in the pro-inflammatory cytokines TNFα and IL-6. These subtle differences in C5a and C5a-des Arg induced receptor function may be of benefit in understanding the regulation of C5a in acute inflammation. [ABSTRACT FROM AUTHOR]

Background The complement cascade is a highly sophisticated network of proteins that are well regulated and directed in response to invading pathogens or tissue injury and forms a major component of our innate immune response. Complement C5a binds to two G-protein coupled receptors (GPCR); namely the C5a receptor (C5a1) and C5a receptor like-2 receptor (C5a2). C5a2 is a non-signalling GPCR and its role has been difficult to validate due to the unavailability of selective ligands. Aims Discover novel C5a2 ligands that allow us to probe and explore the functional role of C5a2 in cytokine release from macrophages. Methods Radioligand binding assays were used to screen a small peptide based library of 61 ligands designed to act at C5a1 for the ability to displace 125 I-C5a from C5a2 expressing membranes. A novel β -arrestin 2 recruitment via C5a2 BRET assay was developed to test for ligand activity at C5a2 and ligands were counter screened for β -arrestin 2 recruitment via C5a1. ELISA was used to measure cytokine release from human monocyte derived macrophages (HMDM). Results Radioligand binding testing of the 61 ligands revealed two ligands (P32 & P59) which showed ⩾ 40% inhibition of 125 I-C5a binding to C5a2 membranes at 100 μ M. P32 and P59 were able to dose dependently recruit β -arrestin 2 via C5a2 but not C5a1 each with an EC 50 of 7.6 μ M. Neither P32 nor P59 was able to recruit β -arrestin 2 to a similar level as C5a, with P32 able to recruit to ∼ 55% and P59 ∼ 30% of the level of C5a. Both P32 and P59 could dose dependently inhibit the release of IL-6 from HMDM when co-stimulated with LPS but had no effect on IL-10 release. Conclusions P32 and P59 are the first reported selective ligands for C5a2 and show novel pharmacology with the ability to modulate cytokine release from HMDM. [ABSTRACT FROM AUTHOR]