Activated neutrophils are responsible for much of the tissue injury in systemic lupus erythematosus (SLE). The primary mediators of neutrophil activation in SLE are complement-derived proteins including C5a, opsonized immune complexes (IC) and the terminal membrane attack complex C5b-9. Ras-related GTP-binding proteins, which require a specific series of posttranslational modifications (prenylation, proteolysis and carboxyl methylation) for membrane targeting and biological activity, have been implicated in a wide variety of signaling pathways. We have recently demonstrated that activation of human neutrophils by the chemoattractant FMLP is associated with increased carboxyl methylation of several ras- related proteins. Activation of one of these GTP-binding proteins, p22rac2, induced its translocation from cytosol to plasma membrane. We also showed that specific inhibitors of ras carboxyl methylation (e.g. N-acetyl-S-farnesylcysteine (AFC) dramatically inhibited neutrophil O2 production. The purpose of this proposal will be to: i. demonstrate signaling through ras proteins in neutrophils activated by C5a, IC and C5b-9; ii. elucidate the signaling pathways regulated by carboxyl methylation of ras proteins and test in vivo an inhibitor of ras methylation for antiinflammatory activity; iii. determine the requirements for membrane translocation of neutrophil p22 rac2 and identify novel prenylcysteine binding proteins. We will test the hypothesis that C5a, IC and C5b-9 signal through ras proteins by determining GTP/GDP ratios, MAP kinase tyrosine phosphorylation and activity and the extent of carboxyl methylation of specific ras-related proteins following activation of neutrophils with a variety of stimuli including C5a, IC and C5b-9. We will also determine if ras-related proteins are associated with C5b-9-enriched shed neutrophil vesicles. We will elucidate ras carboxyl methylation signaling pathways by exposing neutrophils to AFC prior to activation and analyzing neutrophil function and intracellular second messenger generation. We will use the carrageenin-induced inflammation into a murine air pouch as a model of acute inflammation with which to test the in vivo antiinflammatory activity of AFC. We will determine the membrane requirements for cytosolic and recombinant p22rac2 translocation and test the hypothesis that carboxyl methylation is required for translocation by analyzing the subcellular localization of p22rac2 in cytoplasts stimulated in the presence or absence of AFC. We will use photoaffinity labeling and affinity chromatography to identify novel prenylcysteine binding proteins. Elucidation of the mechanisms of ras-mediated neutrophil activation may reveal novel strategies for antiinflammatory therapy in SLE.
