Figure 1.

Neutrophil DREAM plays a positive role in neutrophil recruitment in TNF-α–induced vascular inflammation. (A–F) Intravital microscopy of WT and DREAM KO mice was performed as described in the Materials and methods. Vascular inflammation was induced by intrascrotal injection of TNF-α. 3 h later, rolling, adherent, crawling, and transmigrated neutrophils were visualized in the cremaster venules by infusion of an Alexa Fluor 647–conjugated anti–Ly-6G antibody. The numbers of rolling, adherent, and transmigrated neutrophils were counted in each vessel for 5 min and normalized to the vessel length. (A) Representative images. “0” was set when image capture began on each vessel. Large arrows indicate the direction of blood flow. Colored arrowheads indicate individual rolling neutrophils over 20 s. Scale bar = 10 µm. (B) The rolling number of neutrophils. (C) The cumulative frequency of the rolling velocity of neutrophils. (D and E) The number of adherent neutrophils and the percentage of crawling cells among adherent neutrophils. (F) The number of transmigrated neutrophils in the field of view (0.02 mm²). (G–I) Vascular inflammation was induced by intrascrotal injection of MIP-2. 2 h later, neutrophil recruitment was evaluated as described above. The numbers of rolling (G), adherent (H), and transmigrated neutrophils (I). (J–M) Neutrophil recruitment was induced by superfusion with 10 µM fMLP at 37°C for 20, 40, and 60 min. The first capture before fMLP superfusion is shown as time 0. (J) The rolling speed and the numbers of rolling (K), adherent (L), and transmigrated neutrophils (M) were determined during a 5-min recording period. The horizontal bar represents the median value in B, F, G, and I. Otherwise, data represent the mean ± SD (n = 46–49 venules in seven mice/group for A–F, n = 30 venules in three mice/group for G–I, and n = 3 mice/group for J–M). ****, P < 0.0001 versus WT control after Student’s t test (D, E, H, and J–M) or Mann–Whitney U test (B, C, F, G, and I).