Pneumonia caused by Pseudomonas aeruginosa carries the highest mortality and is the most difficult to eradicate of all the Gram-negative bacterial pneumonias. Nevertheless, the precise way in which P. aeruginosa causes disease in man remains unclear and the propensity to progress to chronic infection remains poorly understood. This proposal is concerned with antigen-specific IgG opsonins and the single bacterial enzyme, Pseudomonas elastase, most active in the proteolysis of these immunoglobulins and other structural proteins important to maintaining the integrity of the lung. Goals of this proposal are to define mechanisms whereby the destructive, necrotic and hemorrhagic lung lesions may arise, and to provide explanations or the recalcitrant response and chronic nature of these infections.
Specific aims of this proposal and the major methods to be applied are: 1. Destructive lung lesion: To explain the observed augmentation of Pseudomonas elastase in the presence of Alpha-1-proteinase inhibitor. By what means does this stimulation of bacterial elastolytic activity occur? Is the ability off Alpha-1-PI to inactivate neutrophil proteolytic activity altered in the presence of Pseudomonas elastase? Affinity and standard chromatography techniques will be employed to purify the elastases of P. aeruginosa and human neutrophils; elastolytic activity is measured with 14C-methyl labeled nuchal ligament elastin; and reciprocal plots of enzyme substrate and velocity of reaction will be used to identify competitive inhibitors. 2. Hemorrhagic lung lesion: To define a dose-response relationship between concentrations of purified Pseudomonas elastase and human endothelial cell injury. Human umbilical vein endothelial cells will be grown to confluency before challenge with the bacterial proteases. Permeability to serum proteins, endothelial detachment and lysis and unmasking of Fc-receptors will be quantitated. 3. Chronic lung lesion: To evaluate the role of IgG opsonins in the intracellular bactericidal process. It is postulated that IgG is critical for optimal intracellular processing by pulmonary macrophages. If this is substantiated, phagocyte membrane potential changes, phago-lysosomal fusion and generation of superoxide anion will be studied in an attempt to define the means by which structural immunoglobulin proteins influence intracellular killing of Pseudomonas aeruginosa.
