Surface PDI regulates ligand-binding activity and clustering of αMβ2 integrin without affecting its conformational change. (A-B) Human neutrophils were pretreated with blocking antibodies (10 μg/mL), followed by incubation with fMLF and Alexa Fluor 488–conjugated FG. The fluorescence intensity of FG on inhibitor-treated neutrophils was normalized to that on control cells (100%, white bar). Data represent mean ± SD (n = 3-6). *P < .05 or **P < .01 vs control IgG after ANOVA and the Dunnett test. #P < .05 vs each antibody alone after the Student t test. (C) Human neutrophils were pretreated with mouse IgG1 or IgG2a (IgG1 or IgG2a), or a blocking antibody against PDI (RL90 and BD34) or activated αM (CBRM1/5), 15 μg/mL, and then incubated with or without fMLF. Binding of the PE-conjugated CBRM1/5 was analyzed by flow cytometry. CBRM1/5 binding to neutrophils treated with control IgG was normalized as 100% (white bar). Data represent mean ± SD (n = 3-4). **P < .01 vs control IgG after the Student t test. (D-E) Confocal microscopy was performed as described in “Materials and methods.” Neutrophils were stimulated with fMLF in the presence of control IgG or a blocking anti-PDI antibody and plated onto ICAM-1 surfaces. Adherent cells were stained with PE-conjugated anti-activated αM (CBRM1/5) (red) and Alexa Fluor 488–conjugated anti-PDI antibodies (green). Representative images are shown as low (left panel) and high (right panel) magnifications (n = 4). Little signal was detected by isotype control IgG (data not shown). Colocalization histograms show the intensity of surface PDI and activated αMβ2 integrin along the white line. Bar = 10 μm. (E) The number of medium- (10-100 pixels) and large-sized (>100 pixels) punctates of activated αMβ2 integrin was quantified in confocal images (n = 25-30 cells in 4 independent experiments). P value was obtained from the Mann-Whitney test.