The inflammatory mediators derived from neutrophils and macrophages involved in the pathogenesis of acute as well as chronic lung injury are incompletely understood. Most studies have looked separately at either protease or oxygen radical (oxidant) involvement. These studies suggest that oxidants are critically involved in many types of acute lung injury and proteases, particularly elastase, in chronic lung injury. However, emerging evidence, particularly from in-vitro studies suggests that interactions between these two potent classes of inflammatory mediators may be critical in the evolution of the tissue injury. This proposal will address in a systematic fashion oxidant and protease interactions and how this relates to the pathogenesis of acute lung injury. In initial studies in-vitro we will identify the spectrum of proteases and oxidants produced by rat neutrophils and macrophages since we are using the rat for the in-vivo studies of lung injury. We are interested in determining if rat leukocytes have the same types of proteases and oxidants as the human cells and in preliminary studies this appears to be the case. Rat neutrophils produce hypochlorous acid (HOCL), have latent metalloenzymes just like the human neutrophil and produce significant amounts of elastase. In other in-vitro studies we will assess oxidant-protease interactions in the destruction of extracellular matrix proteins. Based on preliminary studies it appears as if oxidants can directly affect these components to enhance subsequent proteolysis and we will study in detail how oxidants and proteases interact to enhance the breakdown of these extracellular matrix proteins. In correlative in-vivo studies we will address the question of the role that proteases play in two experimental models of acute lung injury which appear to require oxidants to initiate the tissue injury. Since inhibition by antioxidants appears to be time dependent we will do a complete time course study to determine precisely how long after initiation of the injury that antioxidants can effectively suppress the injury. Evidence of protease involvement in these two models of acute lung injury will be assessed as follows. First, in lung lavage we will look for evidence of free protease activity in the injured animals with preliminary studies revealing that they are present. Secondly, we will be looking for evidence of extracellular matrix proteins in the lavage fluid and specifically whether or not they show evidence of proteolysis. In preliminary studies we find that this appears to be the case. In interventional studies we will determine if antioxidants alter the levels of extracellular matrix products and proteases in the lavage fluid of injured rats. Finally, we will assess directly the role that proteases play by the use of protease inhibitors. This study should provide important information on the importance of protease-oxidant interactions in the pathogenesis of acute lung injury.
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