Pseudopod projection and cell spreading of passive leukocytes in response to fluid shear stress

Abstract

Recent evidence suggests that circulating leukocytes respond to physiological levels of fluid shear stress. This study was designed to examine the shear stress response of individual leukocytes adhering passively to a glass surface. Human leukocytes were exposed to a step fluid shear stress with amplitude between 0.2 and 4 dyn/cm(2) and duration between 1 and 20 min. The response of the cells was determined in the form of projected cell area measurements by high-resolution observation before, during, and after fluid shear application. All cells selected initially had a round morphology. After application of fluid shear many cells projected pseudopodia and spread on the glass surface. The number of leukocytes responding with pseudopod projection and the extent of cell spreading increased with increasing amplitude and duration of fluid shear stress. Pseudopod projection after exposure to a step fluid shear occurs following a delay that is insensitive to the shear stress amplitude and duration. Leukocytes that did not project pseudopodia and spread in response to low shear stress could be shown to respond to a second shear step of higher amplitude. The spreading response requires an intact actin network and activated myosin molecules. Depleting the cell glycocalyx with protease treatment enhances the spreading response in sheared leukocytes. These results indicate that passive leukocytes respond to fluid shear stress with active pseudopod projection and cell spreading. This behavior may contribute to cell spreading on endothelium and other cells as well as to transendothelial migration of leukocytes in the microcirculation.

FIGURE 1

Schematic diagram of a glass adherent cell exposed to shear stress from fluid discharge out of a pipette. The distance between the cell center and pipette tip was maintained at ∼10 μm. The pipette was held at an angle of 30° to the substrate and raised a distance of ∼2 μm above the substrate.

FIGURE 2

Digitized images of a round (A) and spreading (C) cell and the corresponding discrete representations of the cell outline (B and D, respectively).

FIGURE 3

Examples of the response of leukocytes exposed to shear stress of 2 dyn/cm² for 5 min. (A) A responding cell that spread extensively on the substrate. Notice that the cell continued to spread after the shear stress was returned to zero. (B) A cell that failed to show any detectable shear stress response.

FIGURE 4

The number of responding cells as a function of the (A) maximum shear stress for cells exposed to a 5-min shear application, and (B) duration of shear exposure for cells sheared with a maximum shear stress of 2 dyn/cm². The filled bars correspond to the number of responding cells in each group. The open bars correspond to the number of nonresponding cells. The numbers over each bar correspond to the number of observations.

FIGURE 5

The response time of responding cells as a function of the (A) maximum shear stress for cells exposed to a 5-min shear application, and (B) duration of shear exposure for cells sheared with a maximum shear stress of 2 dyn/cm². Solid symbols and error bars correspond to the mean ± SD of the observations (open symbols). In B, (*) indicates statistical significance (p < 0.05) compared to τ = 1 min.

FIGURE 6

The maximum normalized area of responding cells as a function of the (A) maximum shear stress for cells exposed to a 5-min shear application, and (B) duration of shear exposure for cells sheared with a maximum shear stress of 2 dyn/cm². Solid symbols and error bars correspond to the mean ± SD of the observations (open symbols). Mean values were significantly different from control observations. In A, (*) indicates statistical significance (p < 0.05) compared to τ = 1 and 5 dyn/cm². In B, (*) indicates statistical significance compared to t_τ = 1 min; ($) indicates statistical significance (p < 0.05) compared to t_τ = 5 min.

FIGURE 7

(A) Typical example of a leukocyte that failed to respond to a shear of 0.2 dyn/cm² for 5 min but did respond to a second shear application at 2 dyn/cm² for 5 min. (B) The maximum normalized area for cells exposed to a single fluid shear exposure of 2 dyn/cm² for 5 min (1 STEP ONLY) and cells exposed to fluid shear of 0.2 dyn/cm² for 5 min followed by a second shear application of 2 dyn/cm² for 5 min (2 STEPS).

FIGURE 8

Initial projected area before application of shear stress and maximum normalized area with application of fluid shear stress at 2 dyn/cm² for 5 min of leukocytes pretreated with 5 and 17 μg/ml HNE (A and B, respectively), 10 mM sodium azide (C and D, respectively), and 1 and 10 mM cytochalasin D (Cyto D) and 5 and 50 mM ML-7 (E and F, respectively).