The leukocyte response to fluid stress

Abstract

Leukocyte migration from a hemopoietic pool across marrow endothelium requires active pseudopod formation and adhesion. Leukocytes rarely show pseudopod formation while in circulation. At question then is the mechanism that serves to minimize leukocyte pseudopod formation in the circulation. We tested the hypothesis that fluid shear stress acts to prevent pseudopod formation. When individual human leukocytes (neutrophils, monocytes) spreading on glass surfaces in vitro were subjected to fluid shear stress ( approximately 1 dyn/cm2), an instantaneous retraction of pseudopods was observed. Removal of the fluid shear stress in turn led to the return of pseudopod projection and cell spreading. When steady shear stress was prolonged over several minutes, leukocyte swelling occurs together with an enhanced random motion of cytoplasmic granules and a reduction of cytoplasmic stiffness. The response to shear stress could be suppressed by K+ channel blockers and chelation of external Ca2+. In rat mesentery microvessels after occlusion, circulating leukocytes project pseudopods in free suspension or when attached to the endothelium, even though immediately after occlusion only few pseudopods were present. When flow was restored, pseudopods on adhering leukocytes were retracted and then the cells began to roll and detach from the endothelium. In conclusion, plasma shear stress in the circulation serves to reduce pseudopod projection and adhesion of circulating leukocytes and vice versa reduction of shear stress leads to pseudopod projection and spreading of leukocytes on the endothelium.

Figure 1

Schematic of the in vitro experiments for (A) adhesive cells with a single ejecting micropipette, and (B) for nonadhesive cells with a dual micropipette setup. The bottom panels show the shear stress along a midline on the cell surface computed numerically as outlined in Materials and Methods. (A) The micropipette is inclined at 30° to the surface and the tip of the pipette is 5 μm from the center of the cell top surface. The cell diameter is assumed 8 μm, its thickness 2 μm, the internal pipette tip diameter 2 μm, and the centerline velocity of the fluid jet out of the pipette tip 0.74 mm/sec. (B) The pipette (tip diameter, 5 μm) on the left serves to hold the cell with a small aspiration pressure in the focal plane. The micropipette on the right serves to apply a fluid jet with centerline velocity of 1 cm/sec at an internal pipette diameter of 3.5 μm. The distance ΔL between the cell surface and the pipette tip was assumed to be ΔL = 7 μm and cell diameter 7 μm. The normal stress reaches low but non-zero levels at the radial position of ≈6.5 μm; its peak is at position A. In both cases, the stress distribution over the surface of the cell is a linear function of the center line velocity of the fluid jet from the ejecting micropipette, but nonlinear function of cell diameter and distance ΔL. Reduction of ΔL in case B from 7 μm to 5.6 μm (80%) causes a doubling of the shear stress to ≈0.83 dyn/cm² on the cell surface.

Figure 2

Selected frames during the time course of pseudopod formation of a neutrophil in a rat mesentery venule. (A–D) Adhering neutrophil. (A) The leukocyte shape is spherical without pseudopods immediately after occlusion of the vessel with a micropipette; (B and C) leukocyte spreading on the endothelium (EC) by active pseudopod formation during stasis, (D) retraction of pseudopods upon restoration of flow. (E–H) Freely suspended neutrophil. (E) The cell is initially spherical. (F) Pseudopod projection shortly after flow stoppage, and continuation throughout stasis (G and H). The cell is carried away from the observation field upon return of flow.

Figure 3

Time course of pseudopod formation for selected neutrophils and monocytes in rat mesentery venules (20–40 μm in diameter) during occlusion (thin lines) and after return of flow (thick lines). L refers to the maximum length across the cell and L_o refers to the diameter of the cell in its spherical state without pseudopods. All leukocytes showed projecting pseudopods to spread on the endothelium (as shown in Fig. 2) and were rolling out of the observation field after retraction of the pseudopod (L/L_o = 1).

Figure 4

Time course of cell spreading and pseudopod formation of a human neutrophil on an a glass slide (A) before, (B) 30 sec, and (C) 120 sec after application of a fluid jet by a micropipette (arrow) (Fig. 1A). The cell continues to retract its pseudopods during application of the fluid jet, but spreads immediately after removal of the pipette and return of fluid shear stress to near zero levels (D).

Figure 5

(A) Time course of human neutrophil spreading before and after application of a fluid shear stress for a period of 2 min by means of the single micropipette setup (Fig. 1A). L represents the maximum length cross the cell and L_o the diameter of the undeformed spherical cell. Control is without application of shear stress. (B) Time course of human neutrophil spreading in the presence of platelet-activating factor before and after application of shear stress for 2 min by means of the single micropipette setup (Fig. 1A). (C) Rate of pseudopod retraction measured as the slope to the curve L/L_o in the first minute after application of shear stress (A) for different levels of shear stress. There are no significant differences between the retraction rates. ∗, P < 0.05 compared with control.

Figure 6

Random velocity of human neutrophil granules and cell volume before and after application of fluid shear stress (Upper) with the dual micropipette setup (Fig. 1B). The neutrophils are nonadherent and have a spherical shape without pseudopod projection. Control is without application of a shear stress. During application there is a significant swelling of the cell and a significant enhancement of Brownian granule motion. This response is blocked after chelation of Ca²⁺ with EDTA (40 mM).

Figure 7

Light micrograph of a human neutrophil (A) before, (B) 5 min, and (C) 10 min after application of a fluid jet to its cell surface with dual micropipette setup (Fig. 1B). There is swelling of the cell and a review of the video tapes shows a vigorous random motion of the granules in C during shear, in contrast to a slow motion of the granules in unsheared cells (A).

Figure 8

Cell surface entry length (mean ± SD) of human neutrophils into the tip of the holding pipette after aspiration (Fig. 1B, left pipette, internal tip diameter ≈3.5 μm) with a negative pressure of 5,000 dyn/cm². Sheared neutrophils were exposed to a peak shear stress of 0.15 dyn/cm² for a period of ≈2 min prior to the application of the aspiration pressure, while the unsheared cells were left in a quiescent medium before aspiration. The sheared cells were completely drawn into the mouth of the micropipette after aspiration, a phenomenon not observed in control cells. ∗, P < 0.05

Figure 9

Granular random velocity and cell volume of human neutrophils upon exposure to 0.15 dyn/cm² for 5 min with the dual micropipette setup (Fig. 1B). For concentrations of the blockers see Materials and Methods. ∗, P < 0.05 compared with control.