The proposed studies are directed toward delineating the pathogenesis of acute lung injury as well as toward the basic biology of the inflammatory cells involved (e.g. neutrophils). The binding of C5a to a receptor on the surface of these inflammatory cells initiates a multitude of responses, including chemotaxis, secretion of lysosomal enzymes, and oxygen free radical production. Very little is known, particularly at the biochemical and structural level, about the receptor for C5a. We intend to purify the rabbit neutrophil C5a receptor to homogeneity. This will be accomplished by immunoaffinity procedures utilizing monoclonal antibodies reactive with the receptor as well as HPLC techniques. We will also map the binding and activation domains of the human C5a molecule by analyzing our battery of monoclonal anti-human C5a antibodies for their ability to inhibit either 125I-C5a receptor binding or neutrophil chemotaxis. We will incorporate the purified receptor protein into phospholipid vesicles and fuse the vesicles with a receptor-deficient cell (e.g. - melanoma tumor cells) with the intention of conveying C5a responsiveness to the acceptor cell as assayed by both biological responses (i.e. C5a induced chemotaxis) and biochemical activation pathways (e.g. 3H-arachidonate release, phosphoinositide metabolites). We will investigate whether the affinity of the C5a receptor is under the control of the guanine nucleotides and their regulatory proteins, whether the control is reversible and whether the association and/or dissociation rates of binding are affected. We will also investigate whether the molecular basis of alterations in C5a receptor binding affinity can be determined using purified receptor protein incorporated into phospholipid vesicles with and without the addition of the purified guanine regulatory proteins. We will also determine whether the C5a receptor possesses intrinsic protein kinase activity per se or in association with other acceptor proteins as well as which amino acid residues undergo phosphorylation. Lastly, to evaluate the in vivo modulation of the C5a receptor and their role in the pathogenesis of acute lung injury, blood neutrophils recovered from rats after systemic complement activation by cobra venom factor will be serially studied with regard to migratory responses, lysosomal enzyme content, superoxide producing capabilities and receptor binding activity for C5a.
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