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 specific for a synthetic peptide based on the predicted amino-terminal sequence of human C5aR (anti-C5aR(9-29)) is capable of binding to both normal human and transfected cells bearing C5aR. Flow cytometric analysis 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 activities 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 cytokine production (IL-6 and IL-8) from human monocytes in vitro. These results suggest that this site-directed polyclonal antiserum specifically 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 blocking 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 suggest that this antiserum does not interact with the C5a "effector" site but sterically interferes with C5a binding to its receptor by interacting with the extracellular amino-terminal region of the receptor.
Morgan
E L
EL
Department of Immunology, Scripps Research Institute, La Jolla, CA 92037.
Ember
J A
JA
Sanderson
S D
SD
Scholz
W
W
Buchner
R
R
Ye
R D
RD
Hugli
T E
TE
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D000954:Antigens, Surface / Q000276:immunology; D001483:Base Sequence; D002634:Chemotaxis, Leukocyte; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis; D016209:Interleukin-8 / Q000096:biosynthesis; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000502:physiology; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000737:chemistry / Q000276:immunology*
D000906:Antibodies; D000954:Antigens, Surface; D015850:Interleukin-6; D016209:Interleukin-8; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement