Joel Swanson, Ph.D.
Professor,
Department of Microbiology and Immunology

jswan@umich.edu

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 of pathogenic bacteria or vaccine adjuvants. These goals are important not only for understanding macrophage biology, but also for elucidating how microbes cause disease and how vaccines initiate immune responses to infection.

To understand how a macrophage coordinates many cell surface receptors to ingest a particle by phagocytosis, we have developed and applied microscopic methods for imaging signaling molecules inside living cells. We determined that phagocytosis by Fc receptors entails two component activities: pseudopod extension and phagosome closure. Inhibition of the enzyme phosphoinositide 3-kinase (PI3K) allows the first but not the second activity to occur. Consequently, phagocytosis proceeds only halfway around a particle. To determine how these distinct activities are coordinated by PI3K, we are examining the regulation of the actin cytoskeleton during phagocytosis, focusing in particular on the dynamics of Rho- and Arf-family GTPases and membrane phosphoinositides. These studies employ ratiometric fluorescence microscopy of macrophages expressing protein chimeras of cyan fluorescent protein or yellow fluorescent protein. Using quantitative fluorescence resonance energy transfer (FRET) microscopy to measure interactions between fluorescent proteins inside macrophages, we identified discrete stages of signaling that correspond to the distinct stages of phagosome formation. One set of GTPases is active early during phagocytosis, when actin-rich cups of membrane extend over particle surfaces. Increased concentrations of 3' phosphoinositides deactivate some of those GTPases and activate a second set, which regulate the later stages of phagosome closure. This PI3K-dependent signal transition that coordinates Fc receptors may be a general mechanism for coordinating receptor activities in cells.

A long-term objective of our studies is to identify 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 pH and calcium concentrations in phagosomes containing L. monocytogenes showed that LLO-mediated disruption of membranes begins with the formation of very small perforations, which are too small to allow exchange of macromolecules but sufficient to delay fusion of phagosomes with lysosomes. With new microscopic methods for measuring dye leakage across membranes, we recently found that macrophages recruit protein kinase C epsilon to vacuoles that have been perforated by LLO. This indicates the existence of a novel cytoplasmic system for detecting damaged intracellular membranes, a system that could influence inflammation or antigen processing that follows phagocytosis of vaccine formulations. We are currently developing methods to quantify disruption of phagosomes and pinosomes and to identify the signals associated with membrane damage.

Activation of macrophages with interferon- g plus LPS or TNF- a increases their resistance to many pathogens, including L. monocytogenes . We are testing the hypothesis that increased resistance to pathogens in activated macrophages results from accelerated delivery of toxic compounds into phagosomes. Quantitative fluorometric methods are used to analyze endocytic compartment physiology in activated and non-activated macrophages. Reactive oxygen intermediates are localized inside Listeria -containing vacuoles of individual cells, comparing listericidal and nonlistericidal macrophages, as well as macrophages from mice with deletions for components of the nitric oxide or reactive oxygen intermediate biosynthetic pathways. Analyzing conditions directly inside individual phagosomes allows us to measure precisely the timing of bacterial activities in phagosomes and the chemistries of host defense that counter those activities. Thus, by imaging molecular activities essential to macrophage biology, we are explaining how cytoplasm is coordinated for complex tasks.

Representative Publications

Henry, R. M., A. D. Hoppe, N. Joshi and J. A. Swanson. 2004. The uniformity of phagosome maturation in macrophages. J. Cell Biol. 164: 185-194.

Hoppe, A. D. and J. A. Swanson. 2004. Cdc42, Rac1 and Rac2 display distinct patterns of activation during phagocytosis. Mol. Biol. Cell. 15: 3509-3519.

Swanson, J. A. and A. D. Hoppe. 2004. The coordination of signaling during Fc receptor-mediated phagocytosis. J. Leukoc. Biol. 76: 1093-1103.

Henry, R. M., L. Shaughnessy, M. J. Loessner, C. Alberti-Segui, D. E. Higgins and J. A. Swanson. 2006. Cytolysin-dependent delay of vacuole maturation in macrophages infected with Listeria monocytogenes . Cell. Microbiol. 8: 107-119.

Shaughnessy, L., A. D. Hoppe, K. A. Christensen and J. A. Swanson. 2006. Membrane perforations inhibit lysosome fusion by altering pH and calcium in Listeria monocytogenes vacuoles. Cell. Microbiol. 8: 781-792.