Analysis of the interaction of human C5a and C5a des Arg with human monocytes and neutrophils: flow cytometric and chemotaxis studies.
C5a and C5a des Arg are potent complement-derived mediators that bind receptors on peripheral blood leukocytes and tissue-specific cellular elements to elicit and amplify inflammatory and immunomodulatory reactions. To study the interactions of C5a and C5a des Arg with these cells, fluorescein conjugates of these ligands were prepared by a new technique and their binding to monocytes, neutrophils, platelets, and endothelial cells was studied with flow cytometry. Fluoresceinated C5a produced neutrophil myeloperoxidase release and chemotaxis and also bound rabbit anti-C5a antibody much like native anaphylatoxin; likewise, fluoresceinated C5a des Arg demonstrated retention of biologic and antigenic activities. Both fluorescein-conjugated C5a and C5a des Arg bound to monocytes and neutrophils in a concentration-dependent, saturable, and homogeneous manner, but 10- to 15-fold higher concentrations of C5a des Arg were required to attain saturable binding of these leukocytes. Ligand binding was specifically inhibited by native purified human C5a in a concentration-dependent manner, while it was unaffected by C3a or N-formyl-methionyl-leucyl-phenylalanine-lysine. There was no evidence of a C5a receptor-negative subpopulation of monocytes or neutrophils. Moreover, comparative binding experiments with leukocytes from multiple normal volunteers showed that a greater percentage of monocytes than neutrophils bound C5a at less than saturable concentrations of ligand (P less than 0.05, 0.5 to 5.0 nM). A representative half-maximal binding of fluorescein-conjugated C5a (C5a des Arg) binding to monocytes and neutrophils was 1.2 nM (30 nM) and 2.6 nM (68 nM), respectively. In contrast, fluorescein-conjugated C5a did not specifically bind to human platelets or umbilical vein endothelial cells.
Yancey
K B
KB
Department of Dermatology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Lawley
T J
TJ
Dersookian
M
M
Harvath
L
L
D001792:Blood Platelets / Q000378:metabolism; D002634:Chemotaxis, Leukocyte; D003182:Complement C5 / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000502:physiology; D015936:Complement C5a; D015937:Complement C5a, des-Arginine; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006801:Humans; D009000:Monocytes / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism*; D013861:Thiocyanates; D014471:Umbilical Veins
D003182:Complement C5; D015937:Complement C5a, des-Arginine; D005452:Fluoresceins; D013861:Thiocyanates; D015936:Complement C5a; D016650:Fluorescein-5-isothiocyanate