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 ^I25I-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 myoinositol 1, 4, 5-trisphosphate (IP₃) as the second messenger possibly causing the [Ca²⁺]_i increase and the release of N-acetyl-β-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.