In response to a variety of stimuli, human neutrophils undergo a respiratory burst in which oxygen is converted to superoxide (O2-), hydrogen peroxide, hypochlorous acid, and possibly hydroxyl radicals. While these agents generally serve a beneficial role in killing pathogenic microbes, they also can cause serious damage to normal tissues during inflammation. In addition, several human diseases are associated with a failure to regulate appropriately the production of these reactive oxygen derivatives. In the adult respiratory distress syndrome, for example, pulmonary damage is thought to occur because of excessive production of phagocyte oxidants, while in chronic granulomatous disease (CGD), failure to produce these molecules leads to life-threatening infections. The studies proposed are part of a long-range goal to elucidate the biochemical mechanisms that regulate the enzyme responsible for generating reactive oxygen derivatives in phagocytes, NADPH oxidase. A second long-range goal is to develop drugs capable of modulating the level of activity of this enzyme. In clinical situations characterized by excessive inflammation, pharmacologic agents Out down-regulate oxidase activity might provide therapeutic benefit. Conversely, drugs capable of enhancing its activity in such a way that the antimicrobial efficiency of the phagocyte is improved might be of benefit to patients suffering from overwhelming infections or from various types of immunodeficiencies. Several key experimental methods have been developed in this laboratory for studying this problem. A fully soluble cell-free oxidase activation system has been developed, characterized, and optimized. A group of 60 CGD patients who have a variety of different mutations encompassing four of the oxidase components have now been characterized and are regularly available for further studies. Through the use of the fully soluble cell-free system, it has been possible to reconstitute oxidase activity in both the defective membranes and cytosols from these patients using partially purified oxidase components. The following specific aims are designed to build upon this progress and focus on several of the major technical and conceptual deficiencies that currently impede the achievement of the long-range goals of this project: 1) to identify and purify the remaining cytosolic oxidase components and study their intermolecular interactions in unstimulated, primed, and activated normal and CGD neutrophils; 2) to identify and purify remaining membrane oxidase components and study their molecular interactions both within the membrane and with cytosolic components; 3) to determine the role of GTP, low molecular weight GTP-binding proteins, ATP, and lipid second messengers in the regulation of NADPH oxidase activation in the cell-free system. These studies may ultimately lead to new pharmacologic methods for controlling oxygen radical-mediated tissue damage as well as to an improved understanding of the pathophysiology of immune disorders such as CGD.
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