This project seeks to define the events that are responsible for the lipopolysaccharide (LPS) induced lung injury that leads to ARDS in association with gram-negative sepsis. We have elected 1) to focus on gram-negative infection because it is a common antecedent of ARDS, and 2) to focus on lipopolysaccharide (LPS), because it is a well-established component of gram-negative bacteria and plays an important role in the induction of the host response.
Aim 1 seeks to define the role of tyrosine kinases in transduction of the signal for production by mononuclear phagocytes of pro-inflammatory cytokines in response to LPS. It is hypothesized that one or more members of the arc-family of tyrosine kinases are important in this process. The identification of kinases that do play an important role in this process will be addressed by a combined biochemical and molecular biological approach. It is also hypothesized that one or more of the src-family kinases participate in membrane proximal events in LPS-induced signal transduction. This will be addressed by determining which of the events required for expression and secretion of tumor necrosis factor-alpha (TNF-alpha) and IL-1beta are blocked by inhibition of tyrosine kinase function or expression. The third aspect of Aim 1 will apply these observations by testing the effect of prototype tyrosine kinase inhibitors on the production of pro- inflammatory cytokines lung vascular injury and lethality in the rabbit model of LPS induced ARDS described in Aim 4 of Project 6.
Aim 2 seeks to determine the role of interferon-gamma (IFN-gamma) in LPS induced pulmonary vascular injury and lethality using, as a model, transgenic mice. The first aspect of this aim will address the hypothesis that IFN- gamma contributes to ARDS-like lung injury and lethality induced by LPS. This will be addressed in transgenic mice in which expression of the IFN- gamma gene has been perturbed, so as to increase or restrict its expression in response to LPS. Although IFN-gamma is produced both by T cells and by NK cells, data from other systems suggests that the NK cell may be the major source of IFN-gamma in response to acute challenge with bacteria or their products. Thus, the second aspect of this aim addresses the hypothesis that NK cells, rather than T cells are primarily responsible for the production of IFN-gamma in response to LPS. This will be addressed by creating transgenic mice in which expression of IFN- gamma is selectively permuted in T cells or in NK cells. The response to LPS of these mice will be compared to mice with normal or non- selectively perturbed expression of IFN-gamma. These studies will further our understanding of the role of cytokines and the pathways leading to their production in ARDS related to gram-negative infection. The ultimate goal of this project and of the SCOR application in general is to provide insights that will lead to novel therapeutic approaches to the ARDS.
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