The Novel Receptor C5aR2 Is Required for C5a-Mediated Human Mast Cell Adhesion, Migration, and Proinflammatory Mediator Production

Abstract

C5a generated during complement activation possesses proinflammatory and immunoregulatory properties critical for the development and modulation of allergic immune responses. In immune cells, C5a mediates its effects through binding to two G protein–coupled receptors, C5aR1 and C5aR2. Mast cells are key effectors in allergic reactions, and decades of research have suggested that the majority of C5a effects on mast cells are mediated through C5aR1, whereas the expression and function of C5aR2 have not been explored. We demonstrated that the human mast cell line Laboratory of Allergic Diseases 2 (LAD2) expresses surface C5aR2 but not C5aR1, whereas CD34+ cell–derived primary mast cells do not express surface C5aR1 or C5aR2. Stem cell factor and IL-4 upregulated C5aR2 expression on LAD2 cells. Furthermore, C5a caused internalization of LAD2 cell-surface C5aR2. We therefore used LAD2 cells as a model to study C5a/C5aR2-induced biological responses and signaling in human mast cells. We found that whereas C5a was unable to induce degranulation, it stimulated GM-CSF, TNF, CXCL10, and CCL2 production. C5a caused ERK phosphorylation, a signaling molecule important in cytokine and chemokine generation. In addition, C5a stimulated adhesion and chemotaxis of mast cells. Wortmannin, an inhibitor of PI3K, and small interfering RNA against β-arrestin-2 blocked C5a-induced adhesion. Silencing of C5aR2 using lentiviral short hairpin RNA rendered the cells unresponsive to C5a-induced adhesion, chemotaxis, and mediator release, as well as ERK phosphorylation. Overall, this study reveals a novel role for C5aR2 in C5a-mediated activation of mast cells and demonstrates that C5aR2 ligation initiates a β-arrestin-2–, PI3K-, and ERK-dependent signaling pathway in these cells.

The Controversial C5a Receptor C5aR2: Its Role in Health and Disease

“…The expression and localization of C5aR2 is species and cell specific, supported by the C5aRs distribution in different subpopulations of mast cells [55]. In human mast laboratory of allergic disease 2 (LAD2) cell line, C5aR2 localized at the cell surface, while C5aR1 intracellularly.…”

“…CD34 + cell-derived primary mast cells expressed C5aRs intracellularly. In human mast cell line HMC-1, both receptors could be detected on the cell surface [51, 55]. The difference in cellular localization may lead to different ligand responses and thus receptor function.…”

“…This suggests that unknown C5aR2 signaling pathways are still waiting to be discovered. Recently, Pundir and colleagues demonstrated that surface C5aR2 on human mast cell line LAD2 functioned in cell adhesion, migration, and proinflammatory mediators production in response to C5a and C5a des Arg (although weakly), which involved ERK, PI3K, and β -arrestin 2 pathways [55]. …”

“…Human mast cell LAD2 with surface C5aR2 responses to ASP led to degranulation, adhesion, and proinflammatory mediator production in an ERK-dependent manner [55]. In short, C5aR2 may be involved in metabolic inflammation, so-called metaflammation.…”

“…Targeting C5aR2 by RNAi, shRNA, or CRISPR-Cas9 can be another effective tool to silence C5aR2 downstream signaling [14, 25, 55, 96]. There are available reagent kits to downregulate or knock out C5aR2 gene expression.…”