β2-Integrin activation and redistribution on polarized and migrating neutrophils. Neutrophils were preincubated with IC487475, BIRT, nonblocking anti-LFA-1-Alexa 546 (TS2/4), nonblocking anti-Mac-1-PE (ICRF44), or mAb 327C-Alexa 488, as indicated. Cell suspensions were then infused into the parallel-plate flow chamber at a shear stress of 2 dyne/cm2 over a monolayer of IL-1β-activated HUVECs. (A) On neutrophil rolling to arrest, real-time detection of β2-integrin distribution was visualized by sequential collection of phase-contrast and immunofluorescent images every second for 90 seconds using a 60 × oil immersion objective. Micrographs are presented as phase-contrast (gray) and deconvolved immunofluorescence (red) overlays representative of approximately 100 individual neutrophil observations in 4 to 6 separate experiments. (B) Fractional distribution of β2-integrin on polarized neutrophils after 90 seconds was determined by quantitating pixel intensity of the fluorescence signal at the leading projection and uropod. Data are presented as mean ± SEM and are representative of the β2-integrin distribution of approximately 10 cells in 4 to 6 separate experiments. *Significance compared with active β2-integrin and LFA-1 at the leading projection (P < .01). #Significance compared with active CD18 and LFA-1 at the uropod (P < .01).
