Shear-induced capping of L-selectin on the neutrophil surface during centrifugation

Abstract

L-selectin on leukocytes is critical in leukocyte tethering and adhesion to inflamed endothelium and lymphocyte homing to lymphoid organs. The spatial distribution of L-selectin on leukocytes controls cellular adhesive function in hydrodynamic shear. How L-selectin changes its position on the cell membrane remains an open question, but a possible candidate is shear stress encountered on the cell surface. Here we demonstrate shear-induced L-selectin polarization on the membrane during the process of centrifugation of resting neutrophils via immunofluorescent microscopy. It was found that randomly distributed L-selectin on neutrophils moves to a polar cap at one end of the cell after centrifugation (300 x g for 2 min) without inflammatory stimuli. This L-selectin redistribution under shear was predicted by Monte Carlo simulations that show how convection dominates over diffusion, leading to L-selectin cap formation during centrifugation at 280 x g or during leukocyte adhesion to the endothelial wall at 1 dyn/cm(2). Those results point to a role for shear stress in the modulation of L-selectin distribution, and suggest a possible alternate mechanism and reinterpretation of previous in vitro studies of L-selectin mediated adhesion of neutrophils isolated via centrifugation.

Figure 1. Representative micrographs of immunofluorescence-labeled CXCR1 or L-selectin on neutrophils

Immunofluorescent micrographs were taken immediately after labeling (0 hour) with Alexa Fluor 546 conjugated anti-CXCR1 Ab or Qdot605-Leu-8/TQ1 anti-L-selectin Ab, respectively (A,D), after 1 hour under static conditions at 4°C without (B,E) or with (C,F) applying a centrifugation step at 300× g for 2 minutes. A total magnification of 400× was applied on all images and the scale bar on (A) indicates 25 μm.

Figure 2. Centrifugation induces L-selectin localization on neutrophils

Maximal CXCR1 or L-selectin cluster arc angle on each neutrophil at each condition was measured and represented in a histogram. (A,D) t_f = 0 h; (B,E) t_f = 1 h static; (C,F) t_f = 1 h with a centrifugation. Three (A-C) or four (D-F) independent experiments were performed and each bar is expressed with a weighted mean ± standard error of the weighted mean. The number of clusters (G) and the cluster area (H) on a cell were calculated and are expressed with mean ± s.e.m. *, P < 0.05 in unpaired and two-tailed student t-tests.

Figure 2. Centrifugation induces L-selectin localization on neutrophils

Maximal CXCR1 or L-selectin cluster arc angle on each neutrophil at each condition was measured and represented in a histogram. (A,D) t_f = 0 h; (B,E) t_f = 1 h static; (C,F) t_f = 1 h with a centrifugation. Three (A-C) or four (D-F) independent experiments were performed and each bar is expressed with a weighted mean ± standard error of the weighted mean. The number of clusters (G) and the cluster area (H) on a cell were calculated and are expressed with mean ± s.e.m. *, P < 0.05 in unpaired and two-tailed student t-tests.

Figure 3. Monte Carlo simulation of L-selectin surface transport predicts cap formation during centrifugation

(A) Fluid streamlines inside and outside of the neutrophil (modeled as a viscous fluid drop) during sedimentation. (B) 3-D plot showing the initial random distribution of L-selectin receptor in the Monte Carlo simulation of cell sedimentation. (C) Final surface distribution of L-selectin receptor after 50 seconds of sedimentation down 5 cm of test tube length at 280× g. (D) Final surface distribution of L-selectin receptor after 3.9 hours of sedimentation down 5 cm of test tube length at 1× g. Cell sedimentation is directed from top-to-bottom, producing an external flow directed from bottom-to-top when observed from a coordinate system moving with the cell center.

Figure 4. Proposed shear-induced L-selectin cap formation model for E-selectin mediated neutrophil rolling under flow

The E-selectin tether with PSGL-1 is represented as a blue line and the E-selectin tether with L-selectin is represented as a red line. The green shades indicate L-selectin distribution on a cell and the yellow arrows indicate possible sequences of events. In the top diagram, a long-lived E-selectin:PSGL-1 tether results in a cell pause. During the cell pause, shear applied on the cell membrane induces L-selectin cap formation. As the applied shear force continues, the E-selectin:PSGL-1 tether dissociates, followed by rotation of the cell. Rotation allows new multiple adhesive interactions of E-selectin with the concentrated L-selectin cap to firmly arrest the cell. Eventually, localized L-selectin is observed upstream in neutrophil rolling experiments on E-selectin (bottom row).