C3a receptor on dibutyryl-cAMP-differentiated U937 cells and human neutrophils: the human C3a receptor characterized by functional responses and 125I-C3a binding.
The anaphylatoxic peptide C3a is part of a basic immunological defense mechanism, the complement system. Research on the human C3a receptor and signal transduction is hampered by the lack of a suitable human cell or cell line. We screened tumor cell lines and human blood cells for a C3a-dependent increase in cytosolic Ca2+ ([Ca2+]i) and analyzed this reaction in a fura-2/AM fluorescence assay for cells in suspension. U937 cells, when differentiated with dibutyryl-cAMP (Bt2cAMP), and purified human neutrophils reacted in a dose-dependent fashion to C3a and a C3a analogue synthetic peptide. We found complete homologous desensitization of this response and no heterologous desensitization to human C5a. Pertussis toxin totally blocked the increase in [Ca2+]i, indicating the possible involvement of a G-protein. Single-cell analysis by digital imaging fluorescence microscopy indicated that neutrophilic granulocytes responded to C3a. In binding studies with Bt2cAMP-differentiated U937 cells and human granulocytes, the 125I-C3a binding was displaced by C3a, yielding one class of C3a binding sites with dissociation constants (Kd) in the low nanomolar range. We identified myo-inositol 1,4,5-trisphosphate (IP3) as the second messenger possibly causing the [Ca2+]i increase and the release of N-acetyl-beta-D-glucosaminidase as one secretory cell response. By functional and binding studies we demonstrated the expression of the C3a receptor on Bt2-cAMP-differentiated U937 cells and human neutrophils and characterized parts of the C3a signal pathway. Our data support a physiological concept in which C3a might be more important than presently thought.
Klos
A
A
Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Germany.
Bank
S
S
Gietz
C
C
Bautsch
W
W
Köhl
J
J
Burg
M
M
Kretzschmar
T
T
D000118:Acetylglucosaminidase / Q000378:metabolism; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D003994:Bucladesine / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D016177:Macrophage-1 Antigen / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism*; D037342:Pertussis Toxin; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007457:Iodine Radioisotopes; D016177:Macrophage-1 Antigen; D010566:Virulence Factors, Bordetella; D003994:Bucladesine; D015544:Inositol 1,4,5-Trisphosphate; D037342:Pertussis Toxin; D000118:Acetylglucosaminidase