We are trying to understand how circulating cells manage to leave the blood stream and home to distinct organs or tissues where they may have crucial physiologic functions or may cause disease. Despite considerable progress in this field, it is still beyond the reach of even the most sophisticated in vitro methodology to simulate the complex interplay of physical, cellular, biochemical, and other factors that influence blood cell behavior in microvessels. Therefore, we are employing intravital microscopy to study the molecular mechanisms of interactions between blood-borne cells and the intact vascular wall by direct observation of lymphoid and non-lymphoid tissues in anesthetized mice. This is achieved by intra-arterial injection of fluorescently tagged leukocyte subsets from human or murine donors or transfected cell lines. We use a fluorescence video-microscopy system and computer-assisted image analysis to quantitatitate cell behavior in the downstream microvasculature. Using this approach, we have demonstrated that leukocyte homing to most target tissues requires an initial tethering step that leads to rolling in postcapillary venules and is followed by an activation step which, in turn, triggers stationary adhesion and emigration. Each of these steps involves distinct molecular pathways whose unique combination is the reason why certain leukocyte subsets migrate to a particular organ, whereas others don't.

We are now working on dissecting the site-specific adhesion cascades that direct myeloid and lymphoid cells, hematopoietic stem cells, erythrocytes (sickle cells) and platelets to normal and diseased tissues. These include: bone marrow, lymph node, Peyer's patch, gut, striated muscle, skin, bone, liver and bladder. Rotation students may be trained in one (or more) of these models (including microsurgical procedures). A panel of mice is available that are genetically deficient in specific adhesion or signaling pathways and transgenic strains that express green fluorescent protein in distinct lymphocyte subsets.

We have also established intravital multi-photon microscopy techniques to investigate the signals that direct the migration, differentiation, and function of cells within tissues. In particular, we are using this system to examine interactions between antigen specific lymphocytes and professional antigen presenting cells in both physiological and pathological settings. These visualization techniques are supplemented by other experimental approaches including static and flow adhesion assays, flow cytometry, biochemistry, molecular biology, and immunoelectron microscopy that allow us to relate our in vivo observations to circumscribed molecular events.