Glucocorticoid Resistance in Acute Lung Injury

University Of Colorado Denver, Aurora CO

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Abstract

DESCRIPTION (provided by applicant): Persistence of activated neutrophils in the airways is associated with morbidity and morality due to acute lung injury (ALI). Although there are reports of glucocorticoids (GC) improving clinical outcome in ALI, there is great variability in response to GC therapy. The overall objective for this project is to better understand the cellular and molecular basis of neutrophil resistance to GC in patients with severe ALI, and to determine whether neutrophils from such patients are less sensitive to GCs than those from patients with mild ALI or control subjects. The current proposal will use cellular and molecular approaches to study the pathogenesis of GC responsiveness of neutrophils in ALI. The central hypothesis which we will pursue is that neutrophils from patients with severe ALI will have greater cell survival, more tissue damaging activities and cytokine secretion than neutrophils from normal controls or patients with mild ALI and other lung disorders. Neutrophils isolated from patients with severe ALI will be more GC resistant than normal neutrophils or patients with mild ALI. This variability in steroid responsiveness will be reflected in neutrophils of normal volunteers following stimulation with LPS in which a subset will demonstrate GC insensitivity. Furthermore we postulate that GC insensitivity of neutrophils in ALI is due to increased constitutive or cytokine driven expression of GCRb and other transcription factors such as AP-1 and NFk_ that drive the induction of proflammatory genes. Our specific aims are to: define neutrophil phenotypes associated with high or low responsiveness to GCs in peripheral blood neutrophils from normal donors. Studies will examine unstimulated neutrophils or neutrophils stimulated with LPS, or neutrophil stimulatory cytokines such as IL-8 for a) cell death and b) cytokine secretion, in the presence and absence of GCs. (specific aim 1); determine if GC resistance of peripheral blood vs airway neutrophils obtained by bronchoalveolar lavage predicts the intensity of the in vivo pulmonary inflammatory response in normal humans given endotoxin into their lungs or the severity of ALI in patients with ALI (specific aim 2); determine the molecular mechanisms by which LPS or neutrophil stimulatory cytokines alter GC sensitivity by: a) determining whether they increase expression of GCRb, an endogenous inhibitor of GC action, in airway and peripheral blood neutrophils obtained from patients with ALI in comparison with levels in normal and LPS-challenged controls; b) identify SR proteins which interact with the primary transcript to stimulate alternative splicing of the GCR to the beta isoform; c) determining whether increased AP-1 and or NfkB activity characteristic of neutrophils in the inflammatory milieu contributes to steroid resistance via trans-repression of GCRa; d) analysis of GCR influence on p38/MAPK activity e) determining whether increased AP-1 and or NFkB activity characteristic of neutrophils in the inflammatory milieu contributes to steroid resistance via trans-repression of GCRa;and activation of NFkB and f) defining the intracellular location of GCRa and b in neutrophils and determine whether these factors are translocated to the nucleus following dexamethasone (DEX) treatment. (specific aim 3) The elucidation of mechanisms underlying steroid resistance will have important consequences for more objective criteria to diagnose steroid resistance, and the development of novel therapeutic modalities in the treatment of ALI as well as other chronic inflammatory conditions where altered GC responsiveness contributes to persistent tissue inflammation.