Neutrophil sequestration and emigration in the lungs take place primarily in the capillaries. Rolling of neutrophils does not occur at this site, because most capillary segments are narrower than neutrophils. Instead, their biomechanical properties, particularly changes in their deformability, may be an important mechanism of regulating the inflammatory response. Deformability may act to delay or stop neutrophils in the capillaries, allowing adhesion and subsequent events to occur. The proposed studies test the hypothesis that biomechanical properties and adhesive properties of neutrophils interact to regulate their response to inflammatory mediators within the bloodstream or the airways.
Aim 1 investigates the biomechanical properties of neutrophils using both filtration techniques and magnetic twisting cytometry, which measures the apparent stiffness and viscosity of neutrophils. The effect of inflammatory mediators on the biomechanical properties, the mechanisms through which these changes occur include the role of phosphatidylinositides, gelsolin, and actin polymerization, and the interactions between mechanical and adhesive events will be determined.
In Aim 2, the adhesive mechanisms important in neutrophil sequestration and lung injury will be determined, particularly the interaction between L-selectin and CD11/CD18 in pulmonary capillaries where rolling does not occur, the role of L-selectin shedding in modulating the inflammatory response, and the role of ICAM-1.
Aim 3 will determine the mechanism by which complement fragments induce a rapid release of neutrophils from the bone marrow, a major source of neutrophils that sequester in the lungs. These studies test the hypothesis that these mediators induce stiffening and detachment of neutrophils from the bone marrow stroma. The effect of complement fragments on biomechanical properties, F- actin content, shape and location of neutrophils in marrow and the role of adhesion molecules (L-selectin, CD11/CD18, and L-selectin shedding) will be determined.
Aim 4 will investigate biomechanical and adhesive events during neutrophil emigration, particularly the changes in biomechanical properties of endothelial cells as neutrophils migrate between them and the role of VLA-4 and VCAM-1 in neutrophil emigration during pneumonia and peritonitis. These studies will help to understand biomechanical and adhesive events and their interactions during inflammation and will examine the therapeutic potential of altering biomechanical events to modulate the inflammatory response.
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