title: Modulation of C3a activity: internalization of the human C3a receptor and its inhibition by C5a.
abstract: The C3a receptor (C3aR) is expressed on most human peripheral blood leukocytes with the exception of resting lymphocytes, implying a much higher pathophysiological relevance of the anaphylatoxin C3a as a proinflammatory mediator than previously thought. The response to this complement split product must be tightly regulated in situations with sustained complement activation to avoid deleterious effects caused by overactivated inflammatory cells. Receptor internalization, an important control mechanism described for G protein-coupled receptors, was investigated. Using rabbit polyclonal anti-serum directed against the C3aR second extracellular loop, a flow cytometry-based receptor internalization assay was developed. Within minutes of C3a addition to human granulocytes, C3aR almost completely disappeared from the cell surface. C3aR internalization could also be induced by PMA, an activator of protein kinase C. Similarly, monocytes, the human mast cell line HMC-1, and differentiated monocyte/macrophage-like U937-cells exhibited rapid agonist-dependent receptor internalization. Neither C5a nor FMLP stimulated any cross-internalization of the C3aR. On the contrary, costimulation of granulocytes with C5a, but not FMLP, drastically decreased C3aR internalization. This effect could be blocked by a C5aR-neutralizing mAb. HEK293-cells transfected with the C3aR, with or without Galpha16, a pertussis toxin-resistant G protein alpha subunit required for C3aR signal transduction in these cells, did not exhibit agonist-dependent C3aR internalization. Additionally, preincubation with pertussis toxin had no effect on C3a-induced internalization on PMNs. C3aR internalization is a rapid negative control mechanism and is influenced by the C5aR pathway.
authors.affiliation: Institute of Medical Microbiology, Hannover Medical School, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Settmacher
authors.forename: D
authors.initials: D
authors.lastname: Bock
authors.forename: H
authors.initials: H
authors.lastname: Saad
authors.forename: S
authors.initials: S
authors.lastname: Gärtner
authors.forename: C
authors.initials: C
authors.lastname: Rheinheimer
authors.forename: J
authors.initials: J
authors.lastname: Köhl
authors.forename: W
authors.initials: W
authors.lastname: Bautsch
authors.forename: A
authors.initials: A
authors.lastname: Klos
mesh_terms: D000143:Acids; D002021:Buffers; D002454:Cell Differentiation; D002460:Cell Line; D015926:Complement C3a / Q000378:metabolism*; D015936:Complement C5a / Q000494:pharmacology*; D003169:Complement Inactivator Proteins / Q000494:pharmacology*; D004306:Dose-Response Relationship, Immunologic; D005434:Flow Cytometry; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007457:Iodine Radioisotopes; D015479:Leukemia, Myelomonocytic, Acute / Q000378:metabolism; D008024:Ligands; D016177:Macrophage-1 Antigen / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D037342:Pertussis Toxin; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020298:U937 Cells; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D000143:Acids; D002021:Buffers; D003169:Complement Inactivator Proteins; D007457:Iodine Radioisotopes; D008024:Ligands; D016177:Macrophage-1 Antigen; D010566:Virulence Factors, Bordetella; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015926:Complement C3a; D015936:Complement C5a; D007371:Interferon-gamma; D037342:Pertussis Toxin; D013755:Tetradecanoylphorbol Acetate