Accumulation of neutrophils during pulmonary inflammation is a process that is highly regulated at many different steps, including delivery of cells to the capillary, interaction with the endothelium and transmigration of the alveolar wall. Our recent observation that leukocyte volume and deformability are involved in the initial retention in the microvasculature has focussed 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 (only 48%) but doubled its volume (and presumably 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 consequent upon a combination of receptor/ligand engagement and the application of physical forces to the cell. 3. This 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 explored in acute pulmonary inflammation in rabbits. These studies explore a completely new process by which the inflammatory response is controlled, and should contribute to our understanding of fundamental mechanisms regulating the behavior of inflammatory cells.

R01HL050319

1993-09-01

1998-08-31