Neutrophils and macrophages possess specific surface receptors for N-formylated oligopeptides and other mediators of inflammation. Occupancy of the receptor induces chemotactic activity resulting in the accumulation of leukocytes at sites of tissue insult. When an accumulation of these cells occurs in the lung, it plays a causal role in mediating tissue damage associated with disease such as adult respiratory distress syndrome (ARDS), emphysema and pulmonary fibrosis. A primary objective of this proposal is to determine the role of enzymes expressed in the neutrophil plasma membrane in modulating the cellular responses to peptide chemoattractants such as N-formyl peptides and C5a. One of these enzymes has neutral endopeptidase (NEP)-like activity while the other enzyme involved has been identified as an aminopeptidase. Specific peptide and antibody inhibitors of these enzymes will be employed to selectively inhibit the expression of their activity and effects on neutrophil responses such as chemotaxis, motility, superoxide anion production, and elastase release will be evaluated. Preliminary data indicate that inhibitors of NEP selectively block neutrophil chemotaxis toward formyl-peptides suggesting that NEP may play an important role in regulating chemotaxis. Another primary objective is to assess the role of guanine nucleotide regulatory proteins (specifically Gn) as well as cytoskeletal proteins in modulating the responsivity of neutrophils to N-formyl peptides. Based on preliminary experiments, the hypothesis to be tested mandates that the receptor be sequestered away from Gn control by a competing pathway that may involve receptor coupling to the cytoskeleton. Thus, the receptor is removed from the signal transduction pathway and the initial response is terminated. This aspect of the proposed studies will focus on the molecular interactions between the receptor and these regulatory proteins, the relationship of these interactions to measured changes in receptor affinity for attractant and ultimately to the control of cellular responses as a result of these interactions. Finally, the mechanism of receptor internalization and its intracellular fate will be examined. For this purpose, novel biotinylated derivatives of N-formyl peptides synthesized in this laboratory will be used for immunofluorescent and electron microscopic studies. Other useful reagents for these studies will include antibodies to the receptor or any assessory proteins (e.g. Gn and selected cytoskeletal proteins).
| Painter, R G; Aiken, M L (1995) Regulation of N-formyl-methionyl-leucyl-phenylalanine receptor recycling by surface membrane neutral endopeptidase-mediated degradation of ligand. J Leukoc Biol 58:468-76 |
