Phagocytic leukocytes play an important role in the body's defense against pathogenic microorganisms. Directed to a pathogen by chemoattractant factor, these cells engage invading organisms via surface receptors and initiate a phagocytic process mediate by a series of signals that follow the receptor-ligand interaction. While this process eventually destroys most bacteria by creating an acidic microbicidal environment in the phagosome that optimizes the effects of simultaneously-released lytic and oxidative agents, pathogenic mycobacteria can subvert normal events to create a favorable phagosomal milieu. However, the mechanisms controlling the environment, and the ways in which in which destruction-evading mycobacteria circumvent them, are not well understood. We hypothesize that pathogens like Mycobacteria tuberculosis evade this process by modifying the initiation of early, interrelated signals that follow their binding to specific macrophage receptors. Based on our previous work, we plan to study the mechanisms controlling phagosomal and intracellular responses to normally killed versus defense evading stimuli, using flow cytometric (for cells in suspension) and fluorimetric (suspensions or adherent cells) methods, including confocal microscopy (adherent cells). Our methods permit cell- by-cell analysis in real time, enabling us to correlate receptor identify/occupancy with consequent responses, and to identify responding versus non-responding cell sub-populations, an important consideration, since defense evasion may proceed by altering the ratio of these sub- populations. We will use stimuli that induce normal phagocyte responses, and those that will study: 1) the regulation of cytoplasmic and phagosomal pH in polymorphonuclear and mononuclear phagocytes (monocyte- derived and alveolar macrophages), and the role of that regulation in effecting cell function, including destruction of the phagocytized entity; 2) the regulation of phagocyte activation responses to (a) mycobacterial lipoarabanomannans, putative mycobacterial virulence factors, and to (b) virulent and avirulent mycobacteria, via specific receptors involved in their processing; 3) the regulation of phagocyte activation by IgG-opsonized stimuli via Fc-receptor-specific subclasses, and 4) whether disproportionate subpopulation responses exist following each of these stimuli, and how these subpopulations responses may be altered. These studies should permit identification of the mechanisms by which virulent mycobacteria evade normal phagosomal pathogen destruction and possibly permit the design of methods to counteract these defense-evading tactics.
Showing the most recent 10 out of 17 publications
