Expression and function of C5a receptor in mouse microvascular endothelial cells.
The complement-derived anaphylatoxin, C5a, is a potent phlogistic molecule that mediates its effects by binding to C5a receptor (C5aR; CD88). We now demonstrate specific binding of radiolabeled recombinant mouse C5a to mouse dermal microvascular endothelial cells (MDMEC) with a K(d50) of 3.6 nM and to approximately 15,000-20,000 receptors/cell. Recombinant mC5a competed effectively with binding of [(125)I]rmC5a to MDMEC. Enhanced binding of C5a occurred, as well as increased mRNA for C5aR, after in vitro exposure of MDMEC to LPS, IFN-gamma, or IL-6 in a time- and dose-dependent manner. By confocal microscopy, C5aR could be detected on surfaces of MDMEC using anti-C5aR Ab. In vitro expression of macrophage inflammatory protein-2 (MIP-2) and monocyte chemoattractant protein-1 (MCP-1) by MDMEC was also measured. Exposure of MDMEC to C5a or IL-6 did not result in changes in MIP-2 or MCP-1 production, but initial exposure of MDMEC to IL-6, followed by exposure to C5a, resulted in significantly enhanced production of MIP-2 and MCP-1 (but not TNF-alpha and MIP-1alpha). Although LPS or IFN-gamma alone induced some release of MCP-1 and MIP-2, pre-exposure of these monolayers to LPS or IFN-gamma, followed by addition of C5a, resulted in synergistic production of MIP-2 and MCP-1. Following i.v. infusion of LPS into mice, up-regulation of C5aR occurred in the capillary endothelium of mouse lung, as determined by immunostaining. These results support the hypothesis that C5aR expression on MDMEC and on the microvascular endothelium of lung can be up-regulated, suggesting that C5a in the co-presence of additional agonists may mediate pro-inflammatory effects of endothelial cells.
Laudes
Ines J
IJ
Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Chu
Jeffrey C
JC
Huber-Lang
Markus
M
Guo
Ren-Feng
RF
Riedemann
Niels C
NC
Sarma
J Vidya
JV
Mahdi
Fakhri
F
Murphy
Hedwig S
HS
Speyer
Cecilia
C
Lu
Kristina T
KT
Lambris
John D
JD
Zetoune
Firas S
FS
Ward
Peter A
PA
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D001667:Binding, Competitive / Q000276:immunology; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000096:biosynthesis; D054426:Chemokine CXCL2; D018925:Chemokines / Q000096:biosynthesis; D015936:Complement C5a / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000276:immunology* / Q000378:metabolism*; D005434:Flow Cytometry; D019085:Fluorescent Antibody Technique, Direct; D005786:Gene Expression Regulation / Q000276:immunology; D007262:Infusions, Intravenous; D007371:Interferon-gamma / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D007457:Iodine Radioisotopes / Q000378:metabolism; D008070:Lipopolysaccharides / Q000008:administration & dosage / Q000494:pharmacology; D008168:Lung / Q000098:blood supply / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008833:Microcirculation / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D015854:Up-Regulation / Q000276:immunology; D014841:von Willebrand Factor / Q000378:metabolism
D000276:Adjuvants, Immunologic; D015703:Antigens, CD; D018932:Chemokine CCL2; D054426:Chemokine CXCL2; D018925:Chemokines; C404915:Cxcl2 protein, mouse; D015850:Interleukin-6; D007457:Iodine Radioisotopes; D008070:Lipopolysaccharides; D010446:Peptide Fragments; D012333:RNA, Messenger; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D014841:von Willebrand Factor; D015936:Complement C5a; D007371:Interferon-gamma