| Macrophages play an important role in immunity by phagocytosing and killing pathogens. However, some pathogens, such as Listeria monocytogenes (Lm) and Mycobacterium tuberculosis, have evolved mechanisms that enable them to survive within macrophages. In order to kill these organisms, macrophages must be activated by IFN-gamma along with other stimuli such as bacterial products. The aim of this thesis was to characterize the chemistries of activated macrophages, in particular regarding their microbicidal mechanisms directed into phagosomes.;The first set of studies examined the role of Ca2+ in Fc receptor-mediated phagocytosis. Ratiometric imaging of intracellular free Ca2+ showed repeated spikes in intracellular Ca2+ levels upon phagocytosis of IgG-opsonized erythrocytes, and these spikes were more prevalent in activated than non-activated macrophages. Inhibition of Ca2+ spikes, by thapsigargin treatment or prolonged incubation of cells in EGTA, had no measurable effect on particle uptake, phagosome-lysosome fusion, or generation of reactive oxygen intermediates (ROI) or reactive nitrogen intermediates (RNI), indicating Ca2+ spikes were not directly involved in the increased microbicidal activity of activated macrophages.;Next, the chemistries involved in the prevention of vacuolar escape of Lm by activated macrophages were investigated. Experiments using macrophages from gp91phox-/- and NOS2-/- mice, as well as ROI/RNI inhibitors, demonstrated that ROI were required for vacuolar retention of Lm, and that ROI-mediated retention was enhanced by RNI. Live-cell imaging with the fluorogenic probe dihydro-2' ,4,5,6,7,7'-hexafluorofluorescein coupled to BSA (DHFF-BSA) showed rapid generation of oxidative chemistries localized to vacuoles containing Lm. Chemistries that oxidized DHFF-BSA were similar to those that retained Lm in vacuoles, making DHFF-BSA useful for the study of macrophage responses to Lm. DHFF-BSA was oxidized more readily in smaller, less spacious phagosomes, but no difference in phagosomal size was observed between activated and non-activated macrophages. Thus, prevention of vacuolar escape of Lm by macrophages is mediated by the rapid generation of ROI and RNI into vacuoles containing Lm. |
