PDI is required for neutrophil recruitment during TNF-α–induced vascular inflammation. Intravital microscopy was performed as described in “Materials and methods.” Neutrophils were visualized by infusion of an Alexa Fluor 647–conjugated anti–Gr-1 antibody. Six to 8 different inflamed venules were monitored in WT and PDI CKO mice. Then, wtPDI or dmPDI, 100 μg, was infused into PDI CKO mice and neutrophil recruitment was further monitored. (A) Representative images. Small (white and gray) and large arrows show rolling neutrophils and blood flow, respectively. (B-D) The rolling influx (rolling cells per minute) and velocity (micrometers per second) of neutrophils and the number of adherent neutrophils (number per field per 5 minutes) are shown. Data represent mean ± SEM (n = 17-24 venules in 3-4 mice per group). (E) The crawling population of adherent neutrophils was monitored over 5 minutes (n = 80-100 neutrophils in 3-4 mice per group). *P < .05, **P < .01, and ***P < .001 vs WT mice; #P < .05 and ###P < .001 vs PDI CKO mice with or without wtPDI or dmPDI after ANOVA and the Dunnett test. The diameter of microvenules observed was in the range of 33.3 to 45.5 μm and the wall-shear rate in the TNF-α–inflamed cremaster venules was approximately 400 to 650 s⁻¹ as described previously.¹⁹  (F-G) WT and PDI KO neutrophils were stimulated with fMLF in the presence of 50 μg/mL His-tagged wtPDI or dmPDI. Bound PDI was washed with RPMI media or carbonate buffer. Binding of recombinant PDI was analyzed by flow cytometry using a PE-conjugated anti-poly His antibody. The gray histogram represents the fluorescent signal of the anti-poly His antibody on PDI-untreated, stimulated neutrophils. (G) PDI binding is shown as a fold increase by the ratio of the geometric mean intensity value of a PE-conjugated anti-His antibody on PDI-treated vs untreated neutrophils (mean ± SD, n = 3). *P < .05 vs unstimulated neutrophils after the Student t test. (H-I) wtPDI or dmPDI, 100 μg, was infused into PDI CKO mice. Neutrophils, β2 integrin, and PDI were visualized by infusion of Alexa Fluor 647–conjugated anti-Gr-1 (red), Alexa Fluor 488–conjugated anti-β2 (green), and PE-conjugated anti-His antibodies (blue), respectively, into PDI CKO mice. Without recombinant PDI, no fluorescence signal was observed by the PE-conjugated anti-poly His antibody in PDI KO mice (data not shown). White arrows and arrowheads show blood flow and rolling neutrophils, respectively. Representative fluorescence images are shown at different time points following recording (n = 17-18 venules in 3 PDI CKO mice). Neutrophils and β2 (yellow); β2 and PDI (turquoise); neutrophils and PDI (magenta); neutrophils, β2, and PDI (white). (I) Fluorescence intensity of PE-conjugated anti-His antibodies was quantified over 2 minutes.