Analysis of the tissue distribution of the rat C5a receptor and inhibition of C5a-mediated effects through the use of two MoAbs.
The C5-anaphylatoxin C5a is a protein of 74 (human) or 77 (rat) amino-acid residues, respectively, the generation of which may be induced by either the classical and/or the alternative pathways. C5a binds specifically to its receptor (C5aR/CD88) which belongs to the superfamily of G-protein-coupled receptors with seven transmembrane segments. In this study we describe the tissue distribution of the rat C5aR (rC5aR) and the blocking of its ligand by the application of two monoclonal antibodies (MoAbs). The first antibody (MoAb R63) which is directed against the amino-terminal domain Ex1 of the rat C5aR was generated in mice immunized with RBL-2H3 cells which had been stably transfected with the rat C5a receptor gene. Checking the rC5aR expression in various tissues bronchial epithelial cells stained positive only in tissue samples from animals with a mycoplasm infection indicating that the receptor may be induced in this cell type as a consequence of an inflammatory process. Using immunohistochemistry there was no evidence for nonmyeloid expression in the large or small intestine, heart, lung, kidney or liver of the normal rat. The MoAb R63 was found to be a reliable tool for the investigation of the expression of the receptor by FACS analyses or immunohistochemistry. Despite numerous attempts neutralizing antibodies could not be generated against the receptor. Therefore a C5a-ligand neutralizing MoAb was generated against the synthesized carboxyterminal 20mer peptide. This antibody (6-9F) recognized the carboxy terminus of C5a/C5a-FLUOS and prevented its binding at a three-fold molar excess as evidenced by FACS-analyses. It also blocked the C5a-mediated signal transduction as demonstrated by the inhibition of intracellular Ca2+-release (at a 16-fold molar excess) and the release of N-Acetyl-beta-D-glucosaminidase (at a 25-fold molar excess).
Rothermel
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Department of Immunology, Georg-August University of Göttingen, Kreuzbergring 57, D-37075 Göttingen, Germany.
Götze
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Zahn
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Schlaf
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G
D000118:Acetylglucosaminidase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015703:Antigens, CD / Q000032:analysis* / Q000276:immunology; D002118:Calcium / Q000378:metabolism; D015936:Complement C5a / Q000037:antagonists & inhibitors*; D004797:Enzyme-Linked Immunosorbent Assay; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D051381:Rats; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D015936:Complement C5a; D000118:Acetylglucosaminidase; D002118:Calcium