Central to defense against pathogenic microorganisms is the macrophage's ability to internalize fluids and particles by endocytosis, which includes the processes of phagocytosis, receptor-mediated endocytosis, and pinocytosis. This lab uses quantitative fluorometric and microscopic methods to study endocytosis in macrophages. Its principle goals are to delineate the mechanisms and regulation of phagocytosis, and to characterize the intravacuolar environment in the presence and absence of pathogenic bacteria. These goals are important not only for understanding macrophage biology, but also for elucidating basic mechanisms of microbial pathogenesis and innate resistance to infections.

To analyze the mechanism of Fc receptor-mediated phagocytosis, we are developing and applying microscopic methods for imaging signaling molecules inside living macrophages. We recently determined that phagocytosis entails two component activities: pseudopod extension and phagosome closure. Inhibitors of phosphoinositide 3-kinase or myosin allow actin polymerization and pseudopod extension, but inhibit a contractile activity that closes the phagosome; consequently phagocytosis proceeds only halfway around a particle. Completion of phagocytosis requires a constriction of the distal margin of the phagosome. To test further the hypothesis that phagocytosis requires two component signaling activities, we developed a method for synchronizing phagocytosis, which affords good temporal resolution of the process and allows us to examine the biochemistry and morphology of the component activities. With this experimental model, we are examining the regulation of actin polymerization and intracellular free calcium concentrations during phagocytosis, and the contributions of various myosin isoforms to phagosome closure. We are also examining the dynamics of signaling molecules related to phagocytosis, using fluorescently labeled probes in living macrophages.

A long-term objective of our studies is to identify those features of macrophage endocytic compartments that counteract intracellular pathogens. Although post-phagocytic delivery of microbes into macrophage lysosomes typically leads to their degradation, some pathogenic microorganisms survive phagocytosis and evade macrophage defense mechanisms. Listeria monocytogenes is an intracellular pathogen that survives by passing from phagosomes into cytoplasm. It secretes a hemolytic protein, listeriolysin O (LLO), which mediates bacterial passage into cytoplasm. Our measurements of phagosomal pH during infection with L. monocytogenes showed that LLO-mediated perforation of phagosomes occurs optimally at pH 6.0, and requires an acidic environment for perforation. We are presently identifying host and bacterial factors that influence escape of L. monocytogenes from phagosomes, by identifying the compartment permeabilized by L. monocytogenes, and determining how this compartment is altered in activated macrophages.

Activation of macrophages with interferon-gamma plus LPS or TNF-alpha increases resistance to many pathogens, including L. monocytogenes. We are testing the hypothesis that increased resistance to pathogens in activated macrophages results from altered phagosome acidification and progression to lysosomes, plus localized delivery of toxic compounds into late endosome-like phagosomes. Quantitative fluorometric methods are being used to measure endocytic compartment dynamics and physiology in activated and non-activated macrophages. We are measuring rates of phagosome maturation, fusion with lysosomes, and fluid-phase solute recycling. Fluorescence microscopic methods are being used to measure intravacuolar pH and intracellular nitric oxide levels, and to localize reactive oxygen intermediates and peroxynitrite in individual phagosomes. Future studies will compare listericidal and nonlistericidal macrophages, as well as macrophages from mice with induced deletions for components of the nitric oxide or reactive oxygen intermediate biosynthetic pathways. Because these studies provide direct measurements of conditions inside the vacuolar compartments of activated macrophages, their results should improve understanding of host defense mechanisms related to infection by L. monocytogenes as well as other intracellular pathogens.