Recoil and stiffening by adherent leukocytes in response to fluid shear

Abstract

Prolonged exposure to fluid shear stress alters leukocyte functions associated with the immune response. We examined the initial response of freshly isolated human leukocytes to fluid shear stress under high magnification. Adherent leukocytes exhibit a rapid biomechanical response to physiological levels of fluid shear stress. After passive displacement in the direction of a constant fluid shear stress, adherent leukocytes actively recoil back in the opposite direction of the fluid flow. Recoil is observed within seconds of the applied fluid shear stress. Simultaneously, fluid shear stress induces a stiffening of the cell. The immediate cell displacement in response to a step increase in fluid shear stress is greatly attenuated in subsequent steps compared to the initial fluid shear stress step. Recoil is not mediated by actin polymerization-dependent mechanisms, as cytochalasin D had no effect on this early response. However, stiffening was determined in part by an intact actin cytoskeleton. Inhibiting myosin force generation with ML-7 abolished the recoil and stiffening responses, implicating force generation by myosin as an important contributor to the early leukocyte response to fluid shear stress. This initial shear stress response may be particularly important in facilitating leukocyte attachment under sustained fluid shear stress by the flowing blood in the microcirculation.

Figure 1

Digital micrograph of a freshly isolated human leukocyte as used in the current experiments. The cell outline is indicated by a white dashed line, and the computed cell centroid is shown as a white cross. The tip of an adjacent pipette used to apply FSS is just visible to the right of the image with the direction of fluid discharge indicated by the arrow.

Figure 2

Response of human leukocytes to FSS. (A) Time course of FSS used to examine the FSS response of leukocytes. Cells were exposed to two step increases of FSS with a peak value τ_MAX = 2 dyn/cm² and duration of 1 min separated by 1 min. (B) Normalized X-position of the cell centroid X_C shown as a function of time during exposure to FSS. The cell moved primarily in the X-direction, showing an immediate displacement in response to FSS followed by recoil back toward its initial position despite the presence of sustained FSS. (C) Normalized Y-position of the cell centroid shown as a function of time during FSS exposure. (D) Normalized projected cell area shown as a function of time during FSS exposure. Data are the mean and standard deviation of 11 observations.

Figure 3

Stress amplitude dependence of the leukocyte response to fluid shear. (A) Time course of FSS exposure. (B) Leukocyte recoil in a fluid shear field. Normalized X-position of the cell centroid of freshly isolated leukocytes exposed to FSS with peak values on the cell surface of τ_MAX = 1, 2, and 4 dyn/cm². (C) Leukocyte stiffening response. The immediate displacement in response to a step increase in FSS increased with τ_MAX but decreased with subsequent shear stress steps. (*) indicates a statistically significant difference (p < 0.05) in immediate displacement between the first and second steps. ($) indicates a statistically significant difference (p < 0.05) in immediate displacement compared to τ_MAX = 1 dyn/cm². The number of observations, n, is given in the legend.

Figure 4

The extent of active recoil in sustained fluid shear. The active recoil ΔX_C in the X-position of the cell centroid increased with the maximum FSS on the cell surface in the first shear step. The magnitude of active recoil decreased in subsequent fluid shear steps. (*) indicates statistical significance (p < 0.05) in active recoil between the first and second steps. The number of observations, n, at each maximum shear stress is given above the symbol.

Figure 5

(A) Time course of FSS application for a single step increase in fluid discharge from the pipette of 3 min duration. (B) Normalized X-position of the cell centroid X_C shown as a function of time during 3 min exposure to FSS. With longer FSS application, cells eventually recoil back to their initial position and even move toward the pipette. Data are the mean and standard deviation of 10 observations.

Figure 6

The instantaneous displacement of the cell centroid, ΔX_C, in response to a step increase in FSS with a peak value of τ_MAX = 2 dyn/cm². (*) indicates statistical significance (p < 0.05). Data are mean and standard deviation of 17 observations.

Figure 7

Cytoskeletal protein involvement in the active recoil and stiffening responses. (A) Time course of shear stress. (B) Normalized X-position of control cells and cells pretreated with the actin filament disrupting drug cytochalasin D at 1 mM. (C) Normalized X-position of control cells and cells pretreated with the myosin light chain kinase inhibitor ML-7 at 50 μM. The number of observations, n, is given in the legend.