C3A ACTIVATION OF LEUKOCYTES IN INFLAMMATION

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DESCRIPTION (Adapted from Investigator's abstract): Infiltration of leukocytes is a characteristic of both acute and chronic inflammatory diseases. Extensive evidence indicates that neutrophils, monocytes and eosinophils are attracted to sites of inflammation by specific chemotactic factors, including complement factors such as C3a and C5a, where the recruited cells may then be further activated by cellular and/or humoral mediators at local tissue sites. C3a is generated from the third and most abundant component of complement and has been shown to selectively induce chemotaxis and degranulation of eosinophils, but not neutrophils. Our published data indicates that C3a activation of neutrophils is entirely secondary to C3a stimulation of eosinophils (Daffern et al., J. Exp. Med. 181:2129, 1995). Receptors to C3a and C5a are expressed on the surface of many inflammatory cells and these cells are activated through intracellular mechanisms that have yet to be fully characterized or understood. Our preliminary functional studies suggest that C3a induces more unique or different signaling events in eosinophils, and perhaps monocytes, than it does in neutrophils despite the presence of C3a receptors on each of these cell types. Dr. Ye's laboratory has recently isolated the cDNA coding for the C3a receptor (C3aR), a new member of the heptahelical transmembrane class of receptors known as the Rhodopsin family (Roglic et al. BBA, 1305:39, 1996). The cDNA for human C3aR has been shown to encode a G protein-coupled receptor with a unique extracellular loop (-170 amino acids) between the fourth and fifth transmembrane regions. This large extracellular loop structure is a feature observed in no other member of the Rhodopsin family of receptors elucidated to date. We propose that this unusually large extracellular loop is a prime candidate for being the receptor binding/effector site of the intact C3a molecule. We have generated polyclonal antibodies to the large extracellular loop of C3aR and this immunoreagent detects the native cell surface receptor. The bioactive synthetic C3a peptide analogues, that were designed years earlier in our laboratory, now also promise to be valuable tools in mapping the effector binding site on the C3a receptor. Our studies will explore C3a signaling/transduction mechanisms in neutrophils, monocytes and eosinophils, as well as determine characteristics of the C3a/C3aR interactions and mapping of the effector site on the C3a receptor molecule. In this application, we propose a collaborative study to compare the functional responses, binding parameters and molecular mechanisms by which neutrophils, monocytes and eosinophils are differentially activated by the C3a anaphylatoxin.