Analysis of the interaction of human C5a and C5a des arg with human monocytes and neutrophils

Flow cytometric and chemotaxis studies

Kim B. Yancey, Thomas J. Lawley, Mirra Dersookian, Liana Harvath

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

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-leucylphenylalanine- 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 < 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.

Original languageEnglish (US)
Pages (from-to)184-189
Number of pages6
JournalJournal of Investigative Dermatology
Volume92
Issue number2
StatePublished - Feb 1989

Fingerprint

des-Arginine Complement C5a
Chemotaxis
Monocytes
Neutrophils
Fluorescein
Leukocytes
Endothelial cells
Platelets
Ligands
leucyl-phenylalanine
Blood Platelets
Endothelial Cells
Anaphylatoxin C5a Receptor
Anaphylatoxins
Umbilical Veins
Flow cytometry
Peroxidase
Lysine
Anti-Idiotypic Antibodies
Healthy Volunteers

ASJC Scopus subject areas

  • Dermatology

Cite this

Analysis of the interaction of human C5a and C5a des arg with human monocytes and neutrophils : Flow cytometric and chemotaxis studies. / Yancey, Kim B.; Lawley, Thomas J.; Dersookian, Mirra; Harvath, Liana.

In: Journal of Investigative Dermatology, Vol. 92, No. 2, 02.1989, p. 184-189.

Research output: Contribution to journalArticle

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abstract = "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-leucylphenylalanine- 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 < 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.",
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