Figure 3.

Neutrophils fail to accumulate or roll on L-selectin at low shear. (A) Neutrophil accumulation on L-selectin at different shear stresses. Cells were allowed to sediment in the flow stream for 10 sec at 0.25 dyn/cm² on a substrate coated with L-selectin at 2 μg/ml to allow similar number of cells to come into direct contact with the substrate. Shear then was increased abruptly to the indicated values, cells were allowed to accumulate for 20 sec, and the number of adherent cells that accumulated was counted. (B) Tethering of neutrophils after an abrupt increase in shear. Neutrophils were allowed to sediment during flow at 0.25 dyn/cm² on substrates coated with 2 μg/ml L-selectin or 0.5 μg/ml P-selectin and then were subjected to a sharp increase in wall shear stress to 5 dyn/cm². The number of cells in each of three classes of interaction with the substrate were scored as described in Materials and Methods at the low and high shear stresses immediately before and after the increase in shear flow, respectively. New cells arriving from outside the field of view upstream at the high shear stress were ignored. Values are mean of two fields of view. Results in A and B are representative of five experiments. (C) Velocity between each video frame of a representative neutrophil perfused at 0.2 dyn/cm² over the same substrate used in B and then subjected to an increase in shear to 5 dyn/cm² at t = 0.8 sec, marked by the arrow. The cell is nonadherent from 0 to 1 sec and is rollingly adherent after 1 sec. Instantaneous velocities were calculated as in Fig. 2.