Normal chemoattractant activation of Mac-1 in LAD neutrophils is insufficient to trigger adhesion to Mac-1 ligand. (A) PAF- or IL-8–triggered up-regulation of Mac-1 on surface of control or LAD neutrophils. Cell surface Mac-1 on intact or agonist-treated neutrophils was assessed by FACS staining using the Mac-1 specific mAb CBRM1/2. (B) Induction of the activation neoepitope CBRM1/5 on surface Mac-1 of intact and chemoattractant-stimulated neutrophils. In panels A-B, neutrophils were stimulated with either 200 ng/mL IL-8 or 100 nM PAF as outlined in “Materials and methods.” Background staining is indicated by the thin black lines. (C) Arrest of PAF-stimulated neutrophils on fibrinogen (coated at 10 μg/mL) under low physiological shear flow. (i) Control or LAD neutrophils activated by PAF identically as in panels A-B were immediately perfused over fibrinogen at a shear stress of 0.5 dyne/cm.2 The number of leukocytes stably arrested on the substrate upon capture was determined. No transient tethers were observed in any of the indicated settings. Results are given as mean ± range of determinations in 2 fields of view. (ii) Instantaneous velocity profile (upper panel, ▪) of a PAF-stimulated control neutrophil tethered and arrested on the fibrinogen-coated substrate deduced from computerized cell tracking analysis.47 Velocity profile of a resting control neutrophil (⬢) is shown for comparison. Velocity profiles of both PAF-stimulated and resting neutrophils from the LAD patient (lower panel, ▪ and ⬢, respectively) show no velocity drops, suggesting lack of transient adhesive tethers. Profiles are shown for representative neutrophils.
