C3A ACTIVATES REACTIVE OXYGEN RADICAL SPECIES PRODUCTION AND INTRACELLULAR CALCIUM TRANSIENTS IN HUMAN EOSINOPHILS

Whereas C5a is a well-established potent activator of eosinophils, the functional role of C3a in the activation of eosinophils is, so far, p oorly understood. Here, the activation of human eosinophils stimulated with C3a was analyzed and compared to C5a activation. Flow-cytometric al measurements revealed that stimulation of eosinophils by C3a result ed in a transient elevation of the intracellular calcium concentration ([Ca2+](i)) in a dose-dependent manner. In addition, the production o f reactive oxygen radical species (ROS) of eosinophils after C3a and C 5a stimulation was measured by lucigenin-dependent chemiluminescence a nd quantified by superoxide dimutase-inhibitable reduction of ferricyt ochrom C. Half maximal and maximal ROS production in response to C3a w as observed at 50 ng/ml and 1000 ng/ml, respectively, whereas C3a-desA rg was inactive. To ensure that C3a stimulation was not caused by cont amination with C5a, monoclonal antibodies were used to demonstrate the specificity of C3a. The effect of C3a was completely abolished in the presence of monovalent antigen-binding fragments of a functionally bl ocking anti-C3a monoclonal antibody. In addition, blockade of the C5a receptor by the monoclonal anti-C5a receptor antibody S5/1 totally inh ibited the C5a-evoked ROS production, whereas the C3a response in the presence of S5/1 was unaffected. Finally, desensitization experiments revealed a homologous desensitization of C3a after restimulation with C3a. In contrast, no cross-desensitization was observed upon stimulati on with C5a. Furthermore, the C3a- and C5a-induced production of ROS o f eosinophils was totally inhibited by pertussis toxin, indicating the involvement of guanine nucleotide-binding proteins (Gi-proteins). In summary, these results demonstrate that C3a is a potent activator for eosinophils initiating transient [Ca2+](i) changes and production of r eactive oxygen species. C3a therefore may play a part in the pathophys iology of diseases with eosinophil and complement activation.