Complement protein 5a (C5a) is a 74-residue anaphylatoxin protein that activates the cell surface G protein-coupled receptor called C5a receptor 1 (C5aR1). Here we investigate the mechanism of C5aR1-activated chemotaxis of human monocyte-derived macrophages (HMDMs) and their secretion of inflammatory chemokines.
Using recombinant human C5a protein, we find that activation of C5aR1 recruits b-arrestin2 via trafficking of a specific Ras-related protein called Rab5a. This in turn activates downstream phosphatidylinositol 3-kinase (PI3K)/Akt signalling that culminates in chemotaxis and secretion of inflammatory chemokines from HMDMs. High-resolution lattice light sheet microscopy on live cells showed that C5a activates C5aR1-GFP internalization and colocalization with Rab5a-tdTtomato, but not with dominant negative mutant Rab5a-S34N-tdTtomato in HEK293 cells.
Further, we showed that Rab5a is significantly upregulated in differentiated HMDMs. Internalization and formation of endocytic vesicles were observed when stimulating with the fluorescent peptide FITC-FKP(dCha)Cha(dR)-OH, a hexapeptide agonist of C5aR1. This could be blocked by the Ac-(2,6-cyclo)-F[OP(dCha)WR], a C5aR1 antagonist. Using fluorescence microscopy in conjunction with protein, peptide agonist and cyclic antagonist labelled with fluorescein, we were able to monitor agonist and receptor internalization, as well as formation of endocytic vesicles in HMDMs. Interestingly, while knockdown of Rab5a inhibited C5aR1-mediated Akt phosphorylation, it did not affect C5aR1-mediated ERK1/2 phosphorylation or intracellular calcium mobilization in HMDMs.
Functional analysis using transwell migration and µ-slide chemotaxis assays indicated that Rab5a regulates C5a-induced chemotaxis of HMDMs. Further, C5aR1 was found to mediate interaction of Rab5a with b-arrestin2, but not with G proteins in HMDMs. C5a protein-induced secretion of proinflammatory chemokines (CCL2, CCL3) from HMDMs was attenuated by Rab5a or b-arrestin2 knockdown, or by pharmacological inhibition with cyclic peptide C5aR1 antagonist, PMX53, or a PI3K inhibitor. These findings reveal a C5a-C5aR1-b-arrestin2-Rab5a-PI3K signalling pathway that regulates chemotaxis and proinflammatory chemokine secretion in HMDMs and suggest new ways of selectively modulating C5aR1-activated inflammatory outputs.