Stimulation of neutrophils by lipopoly-saccharide (LPS) is central to the pathogenesis of sepsis and the Adult Respiratory Distress Syndrome. LPS binds to CD14, a cell surface protein, resulting in actin assembly and adhesion. The intracellular signaling pathway that links the LPS binding of CD14 with functional cellular responses is largely unknown. Based on studies of chemoattractant stimulation of neutrophils and signaling events in mammalian cell lines, a framework now exists to study intracellular signal transduction in human neutrophils by LPS. Several families of intracellular proteins are present in mammalian cells that function to phosphorylate other proteins. These kinases are inactive until phosphorylated, and then are capable of phosphorylating and activating a specific """"""""downstream"""""""" kinase in turn. The applicants hypothesize that following LPS binding to CD14, one or more nonreceptor protein tyrosine kinases (NRPTKs) are activated. Through a series of sequential phosphorylation reactions the signal is passed through a branch of the mitogen-activated protein (MAP) kinase cascade. A member of the MAP/ERK kinase kinase (MEKK) family is activated, which in turn phosphorylates and activates a member of the MAP/ERK kinase (MEK) family which then phosphorylates and activates a member of the MAP kinase family. They have recently reported that the MAP kinase activated in human neutrophils in response to LPS is p38 MAP kinase. In this proposal members of the MEK, MEKK, and NRPTK-families utilized by the neutrophil in response to LPS stimulation will be identified. These kinases will be isolated by anion-exchange chromatography, assayed for activation in response to LPS, and identified by specific antibodies and protein sequencing. Simultaneously, these signaling events will be explored in murine neutrophils. Signaling proteins share a high degree of homology in all mammalian cells, and it is expected that signaling mechanisms utilized by murine and human neutrophils will be nearly identical. The link between LPS binding of CD14, intracellular signaling, and subsequent functional responses will then be established through the use of a specific inhibitor to p38 MAP kinase, genetically modified mice strains, and murine models of pulmonary neutrophil accumulation. The comprehensive study of this signal transduction pathway may lead to new approaches for modifying LPS induced inflammatory responses.
| Nick, Jerry A; Young, Scott K; Arndt, Patrick G et al. (2002) Selective suppression of neutrophil accumulation in ongoing pulmonary inflammation by systemic inhibition of p38 mitogen-activated protein kinase. J Immunol 169:5260-9 |
| Nick, J A; Young, S K; Brown, K K et al. (2000) Role of p38 mitogen-activated protein kinase in a murine model of pulmonary inflammation. J Immunol 164:2151-9 |
| Nick, J A; Avdi, N J; Young, S K et al. (1999) Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils. J Clin Invest 103:851-8 |
