Mechanical deformation of neutrophil into pulmonary capillaries induces cytoskeletal remodeling, pseudopod projection and changes in biomechanical properties

Abstract

Neutrophils traversing the pulmonary microcirculation are subjected to mechanical stimulation during their deformation into narrow capillaries. To better understand the time- dependant changes caused by this mechanical stimulus, in the first part of the thesis, neutrophils were caused to flow into a microchannel, which allowed simultaneous visualization of cell morphology, and passive rheological measurement by tracking the Brownian motion of endogenous granules. Above a threshold stimulus, mechanical deformation resulted in neutrophil activation with pseudopod projection. The activation time was inversely correlated to the rate of mechanical deformation experienced by the neutrophils. A reduction in shear moduli was observed within seconds after the onset of the mechanical stimulus, suggesting a sudden disruption of the neutrophil cytoskeleton when subjected to mechanical deformation. However, the magnitude of the reduction in moduli was independent of the degree of deformation. Recovery to nearly the initial values of viscoelastic moduli occurred within one minute. These observations confirm that mechanical deformation of neutrophils, similar to conditions encountered in the pulmonary capillaries is not a passive event; rather, it is capable of activating the neutrophils and enhancing their migratory tendencies. The second part of the thesis seeks to understand the changes in the cytoskeletal structure and the extent of biological activation as a result of this deformation process. Neutrophils were passed through narrow polycarbonate filter pores under physiological driving pressures, fixed and stained downstream in order to visualize the F-actin content and distribution.

(cont.) Below a threshold capillary size, the cell remodeled its cytoskeleton through initial F-actin depolymerization, followed by recovery and increase in F-actin content associated with formation of pseudopods. 'This rapid depolymerization and subsequent recovery of F-actin was consistent with our previous observation of an immediate reduction in moduli with eventual recovery when the cells were subjected to deformation. Results also show that neutrophils must be retained in their elongated shape for an extended period of time for pseudopod formation, suggesting that a combination of low driving pressures and small capillary diameters promotes cellular activation. These observations show that mechanical deformation of neutrophils into narrow pulmonary capillaries have the ability to influence cytoskeletal structure, the degree of cellular activation and migrational capabilities of the cells.