The Novel Receptor C5aR2 Is Required for C5a-Mediated Human Mast Cell Adhesion, Migration, and Proinflammatory Mediator Production.
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.
Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island C1A 4P3, Canada;
Priyanka
P
Pundir
National Institute for Nanotechnology, National Research Council Canada, Edmonton, Alberta T6G 2M9, Canada; and.
Clayton A
CA
MacDonald
National Institute for Nanotechnology, National Research Council Canada, Edmonton, Alberta T6G 2M9, Canada; and Department of Medical Microbiology and Immunology, Faculty of Medicine, University of Alberta, Edmonton, Alberta T6G 2E1, Canada marianna.kulka@nrc-cnrc.gc.ca.
Marianna
M
Kulka
D000730:Androstadienes / Q000494:pharmacology; D018952:Antigens, CD34 / Q000378:metabolism; D019390:Arrestins / Q000235:genetics; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology; D015550:Cell Degranulation / Q000276:immunology; D002460:Cell Line; D002465:Cell Movement / Q000276:immunology*; D018932:Chemokine CCL2 / Q000096:biosynthesis; D054357:Chemokine CXCL10 / Q000096:biosynthesis; D015936:Complement C5a / Q000276:immunology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005347:Fibroblasts; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis; D006801:Humans; D007249:Inflammation / Q000276:immunology*; D007371:Interferon-gamma / Q000378:metabolism; D015847:Interleukin-4 / Q000378:metabolism; D008407:Mast Cells / Q000276:immunology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering; D044087:Receptor, Anaphylatoxin C5a / Q000378:metabolism; D019707:Receptors, Chemokine / Q000235:genetics / Q000378:metabolism*; D019089:Stem Cell Factor / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D000077191:Wortmannin; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605254:ARRB2 protein, human; D000730:Androstadienes; D018952:Antigens, CD34; D019390:Arrestins; C488033:C5AR1 protein, human; C417003:C5aR2 protein, human; C111835:CCL2 protein, human; C114753:CXCL10 protein, human; D018932:Chemokine CCL2; D054357:Chemokine CXCL10; C508598:IL4 protein, human; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D044087:Receptor, Anaphylatoxin C5a; D019707:Receptors, Chemokine; D019089:Stem Cell Factor; C439524:TNF protein, human; D014409:Tumor Necrosis Factor-alpha; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D015847:Interleukin-4; D015936:Complement C5a; D007371:Interferon-gamma; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D048049:Extracellular Signal-Regulated MAP Kinases; D000077191:Wortmannin