ANTI-C5A RECEPTOR ANTIBODIES - CHARACTERIZATION OF NEUTRALIZING ANTIBODIES SPECIFIC FOR A PEPTIDE, C5AR-(9-29), DERIVED FROM THE PREDICTED AMINO-TERMINAL SEQUENCE OF THE HUMAN C5A RECEPTOR

Results obtained indicate that a site-directed polyclonal antibody spe cific for a synthetic peptide based on the predicted amino-terminal se quence of human C5aR (anti-C5aR(9-29)) is capable of binding to both n ormal human and transfected cells bearing C5aR. Flow cytometric analys is of stable murine L cell transfectants (C5aR-neo), human neutrophils , and human monocytes indicated that these cells bound anti-C5aR(9-29) in a specific manner. Moreover, F(ab')2 fragments of anti-C5aR(9-29) specifically neutralized proinflammatory and immunoregulatory activiti es induced by natural human C5a. This antiserum was found to block, in a dose-dependent manner, 1) zymosan-induced neutrophil chemotaxis, 2) C5a-induced enzyme release from neutrophils, and 3) C5a-induced cytok ine production (IL-6 and IL-8) from human monocytes in vitro. These re sults suggest that this site-directed polyclonal antiserum specificall y interacts with the human C5aR molecule. To the best of our knowledge , none of the existing reports in the literature provided evidence for a site-directed antiserum to C5aR that was capable of specifically bl ocking C5a-mediated inflammatory/immunoregulatory activities in vitro. Studies conducted with anti-C5aR(9-29) indicated that this antiserum effectively blocked C5a-mediated cell activation but by itself did not activate either neutrophils or monocytes. Combined, these data sugges t that this antiserum does not interact with the C5a ''effector'' site but sterically interferes with C5a binding to its receptor by interac ting with the extracellular amino-terminal region of the receptor.