Accumulation of neutrophils in the pulmonary airspaces, as in ARDS, is a process that is highly regulated at many different steps, including delivery of cells to the pulmonary capillary, interaction with the endothelium and transmigration of the alveolar wall. Our recent suggestion that leukocyte colume and deformability are involved in the initial retention in the microvasculature has focused attention on the role of cell volume in regulating other inflammatory functions of the cell. Thus the relatively small circulating neutrophil was found to have an extremely low water content (less than 50%) but doubled its volume (and presumable its water content) during migration into the inflamed lung. From observations such as these we derived the following hypotheses: 1. The circulating, small, dry neutrophil is in a state of functional latency. 2. Migration initiates an increase in cell volume and water content that involves activation (by phosphorylation) of the Na+/H+ antiport. This is suggested to result from a combination of receptor/ligand engagement and the application of physical forces to the cell. 3. The increase in volume and water content now allows optimal cell functions, including migration (particularly through three-dimensional matrices such as the interstices of the lung), phagocytosis and the production of new gene products including inflammatory cytokines and anti-oxidants. These hypotheses will be examined using a quantitative in vitro migration system in which the volume of neutrophils migrating through a collagen matrix will be determined by 3-D reconstruction using confocal microscopy. The volume and water content of neutrophils will be altered osmotically and by activation or inhibition of the Na+/H+ antiport as well as in the migration system to examine the role of increased water in facilitating migration and phagocytosis and the relationship of phosphorylation of the Na+/H+ antiport and cytoskeletal proteins to these processes. Volume regulation of neutrophil transcription will be studied by PCR, in situ hybridization and detection of specific proteins. The alterations in cell volume and function will then be examined in acute pulmonary inflammation in rabbits and rats an in patients with ARDS. These studies explore a completely new mechanism by which the pulmonary inflammatory response is regulated, and should contribute to our understanding of fundamental mechanisms regulating the behavior of inflammatory cells.
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