Towards a Molecular Signature of Neutrophil Priming

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DESCRIPTION (provided by applicant): Neutrophil priming is an integral aspect of a well-regulated inflammatory response and is the first step in controlling microbial infection and repair of injury. In this model, an initial injury "primes" neutrophils such that a secondary insult, which would otherwise be tempered in an unprimed cell, now results in an exaggerated inflammatory response. The excessive and unregulated release of cytokines and toxic oxygen radicals causes damage to adjacent tissues leading to tissue dysfunction, organ failure and death. The precise molecular pathways initiated upon priming of human neutrophils are poorly understood. The present proposal focuses on the application of quantitative phosphoproteomic technology to understand the unique proteome-wide tyrosine phosphorylation events associated with human neutrophil priming by exposure to low dose N-formyl-met-leu-phe, lipopolysaccharide or platelet activating factor. Using currently available quantitative phosphoproteomic technology and expertise with primary human neutrophils in our laboratories, we will sample primed neutrophils at various time points and quantify relative changes in wide-scale tyrosine phosphorylation from total cell lysates of cellular peptides. Information to be derived from this project include: global identification of tyrosine phosphorylation events that occur over time after neutrophil priming; visualization of the network of signal transduction pathways that are activated in the primed neutrophils; comparison of activation events that take place over time from neutrophils primed with different proinflammatory mediators. This type of quantitative analysis of global tyrosine phosphorylation sites during a time course of neutrophil priming along with traditional biochemical analysis will permit the construction of a signaling pathway network model for neutrophil priming. Together these findings will test the hypothesis that the primed state of the neutrophil includes signaling events that are common among proinflammatory mediators and can be presented as a molecular signature of this fundamental cellular response. Given that responding neutrophils must interpret a number of signals at a site of inflammation, the results from these studies may assist in the development of novel diagnostic or therapeutic reagents aimed at limiting the auto-inflammatory tissue damage patients suffer as a result of sepsis and trauma. PUBLIC HEALTH RELEVANCE: Neutrophil priming is an integral aspect of a well-regulated inflammatory response and is the first step in controlling microbial infection and repair of injury. The precise molecular pathways initiated upon priming of human neutrophils are poorly understood. In this proposal we introduce modern methods in quantitative mass spectrometry to facilitate the characterization of the cellular signaling pathways initiated upon neutrophil priming by a variety of agents by providing a global view of the phosphorylation state of normal and primed cells.