Surface PDI regulates αMβ2-mediated neutrophil adhesion under shear and static conditions. (A-B) Confluent HUVECs on FG-coated glass coverslips were stimulated with TNF-α (20 ng/mL) and placed into a flow chamber. Human neutrophils, 3 × 10⁶, were pretreated with blocking antibodies and stimulated with fMLF. (B) Anti-PDI (BD34, 30 μg/mL) and either anti-αM (15 μg/mL) or anti-αL (50 μg/mL) antibodies were coincubated. Neutrophils were perfused for 10 minutes over activated HUVECs under venous shear of 1 dyne/cm². Then, the medium was perfused for 5 minutes to wash out weakly bound cells. Adherent neutrophils were monitored in a field of 0.15 mm² and counted in 5 to 7 separate fields. (C) Human neutrophils were incubated with anti-PDI (30 μg/mL), anti-αM (10 μg/mL), and anti-αL antibodies (30 μg/mL). Cells were plated onto immobilized ICAM-1 in the presence of fMLF. (D) WT or PDI KO mouse neutrophils were treated with antibodies and plated onto immobilized mouse ICAM-1 in the presence of fMLF. (B-D) The number of adherent neutrophils treated with an inhibitor was normalized to that of adherent neutrophils treated with control IgG (100%). Data represent mean ± SD (n = 3-4 for human neutrophils and n = 6 WT and 6 PDI CKO mice). *P < .05, **P < .01, or ***P < .001 vs control IgG after ANOVA and the Dunnett test. #P < .05 and ##P < .01 vs either anti-PDI + anti-αL or anti-αL + anti-αM antibodies after ANOVA and the Dunnett test (for human neutrophils) or vs WT control after the Student t test (for mouse neutrophils). Round and spread neutrophils are shown as open and filled bars, respectively. Statistical significance was determined by comparison of the number of total adherent (round and spread) cells.