C3A RECEPTOR ON DIBUTYRYL-CAMP-DIFFERENTIATED U937 CELLS AND HUMAN NEUTROPHILS - THE HUMAN C3A RECEPTOR CHARACTERIZED BY FUNCTIONAL-RESPONSES AND I-125 C3A BINDING

被引:77
作者
KLOS, A
BANK, S
GIETZ, C
BAUTSCH, W
KOHL, J
BURG, M
KRETZSCHMAR, T
机构
[1] Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Konstanty-Gutschow-Strasse 8, D-3000 61, Hannover
关键词
D O I
10.1021/bi00161a003
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 I-125-C3a binding was displaced by C3a, yielding one class of C3a binding sites with dissociation constants (K(d)) 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.
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页码:11274 / 11282
页数:9
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