Various clinical syndromes have been shown to possess a common set of pathophysiologic events that appear to be driven in part by lipopolysaccharide activation of the immune/inflammatory system. These maladies may include: thermal injury, multiple trauma, and sepsis. In many of these cases, the lung is one of many target organs involved in the pathology induced by exaggerated host cell activation events. In order to elucidate factors that dictate the induction of these events, this proposal will examine the cellular production and mechanism that regulate gene expression by various macrophage populations. Interleukin-1 (IL-1), tumor necrosis factor (THF-alpha), and transforming growth factor-beta (TGF-beta) will be analyzed and examined for their production and regulation at the cellular and molecular levels. The modulation of these cell-to-cell communication signals by alveolar macrophages and Kupffer cells will be assessed at the antigenic, bioactive, and mRNA levels. The in vivo production of the above mediators will also be studied in the ascites fluid and serum of complete Freund's adjuvant immunized mice challenged with graded doses of LPS. In addition, the kinetics of mRNA accumulation and regulation by macrophages from treated animals will be examined. Initial in vivo studies will assess the ability of TNF-alpha, IL-1alpha, and IL-1beta to act alone or in concert with each other to mimic LPS-induced organ pathology. Techniques and methodologies employed throughout this proposal include: bioassays for IL-1, TNF, TGF-beta; immunohistochemical and Western blot analyses; Northern blot, nuclear transcription, mRNA stability studies and in situ hybridization analysis for the molecular biology studies; and histologic and permeability assessment of organ injury. Unique aspects of this proposal are the implementation of molecular approaches to study monokine production and regulatory mechanism of gene expression by macrophages during cell activation. These techniques will be used to monitor therapeutic approaches to aid our understanding and treatment of LPS-mediated disease processes. This study will provide important knowledge regarding 1) the molecular mechanisms of IL-, TGF-beta, and TNF production and regulation during macrophage activation; 2) cellular and molecular parameters which preface subsequent LPS-induced injury; and 3) mechanisms and efficacy for pharmacologic manipulations that may regulate LPS-mediated tissue injury.
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