Rothermel E, Götze O, Zahn S, Schlaf G
Department of Immunology, Georg-August University of Göttingen, Kreuzbergring 57, D-37075 Göttingen, Germany.
Scand J Immunol. 2000 Oct;52(4):401-10. doi: 10.1046/j.1365-3083.2000.00795.x.
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).
C5过敏毒素C5a是一种分别由74个（人类）或77个（大鼠）氨基酸残基组成的蛋白质，其产生可由经典途径和/或替代途径诱导。C5a特异性结合其受体（C5aR/CD88），该受体属于具有七个跨膜区段的G蛋白偶联受体超家族。在本研究中，我们描述了大鼠C5aR（rC5aR）的组织分布以及通过应用两种单克隆抗体（MoAb）对其配体的阻断作用。第一种抗体（MoAb R63）针对大鼠C5aR的氨基末端结构域Ex1，在用稳定转染了大鼠C5a受体基因的RBL-2H3细胞免疫的小鼠中产生。检查各种组织中的rC5aR表达，支气管上皮细胞仅在支原体感染动物的组织样本中呈阳性染色，表明该受体可能由于炎症过程而在这种细胞类型中被诱导。使用免疫组织化学方法，在正常大鼠的大肠、小肠、心脏、肺、肾脏或肝脏中没有发现非髓样表达的证据。发现MoAb R63是通过流式细胞术分析或免疫组织化学研究该受体表达的可靠工具。尽管进行了多次尝试，但未能产生针对该受体的中和抗体。因此，针对合成的羧基末端20聚体肽产生了一种C5a配体中和MoAb。该抗体（6-9F）识别C5a/C5a-FLUOS的羧基末端，并以三倍摩尔过量阻止其结合，流式细胞术分析证明了这一点。它还阻断了C5a介导的信号转导，如细胞内Ca2+释放的抑制（在16倍摩尔过量时）和N-乙酰-β-D-氨基葡萄糖苷酶的释放（在25倍摩尔过量时）所证明。
