FIGURE 8.

Integrin redistribution and PMN polarization is disrupted by inhibiting SFK. Neutrophils pretreated with SFK inhibitor or vehicle control were labeled with 20 mg/ml anti-CD11a (TS2/4) or anti-high-affinity CD18 (327C) conjugated to Alexa 546 fluorophore for 10 min at room temperature. Neutrophils were then perfused over IL-1β stimulated HUVEC monolayers at 2 dyne/cm² shear stress. Sequences of brightfield and Alexa 546 emission were captured from random fields of arrested PMN from 3 to 6 min following the onset of perfusion. a, Images of LFA-1 or active CD18 are representative of >30 neutrophils and were enhanced by both gamma adjustment to identify cluster location and traced to clarify the neutrophil border. Arrows indicate the location of high-density integrin clusters. b, Polarized PMN that expressed a cluster within 1 μm of the uropod, and at least one additional distinct cluster >3 μm from the uropod, were defined as having biaxial integrin distribution (n = 30–35 neutrophils from three independent experiments; +, p < 0.05).