I-domain allosteric regulation of LFA-1 extension during neutrophil activation. (A-B) Isolated human neutrophils were preincubated with 1 μM IC487475, 1 μM BIRT or vehicle, and the CD18 extension reporter mAb KIM127. Suspensions were then activated with 1 nM IL-8 and were incubated with FITC-conjugated secondary goat anti-mouse F(ab′)2 before fixation and analysis by flow cytometry. Fluorescence histograms (A) are representative of the average population response from 6 separate experiments. Bar graph data (B) represent the mean ± SEM from 6 separate experiments. *Significance in KIM127 binding between BIRT and both IC487475 (P < .01) and vehicle-treated PMNs (P < .05). (C) rLFA-1 heterodimer was derivatized to latex microspheres and incubated with allosteric inhibitors IC487475, BIRT, and lovastatin. rLFA-1 beads were then incubated with MgCl2 and mAb R3.1, followed by secondary labeling with fluorescent goat anti-mouse F(ab′)2 and flow cytometry. Data are given as mean ± SEM for 4 separate experiments and are presented as percentage inhibition relative to maximal binding by vehicle-treated rLFA-1 beads. *Significant inhibition of R3.1 binding compared with vehicle (P < .001).
