P-Rex 1 is required for Mac-1 dependent intravascular crawling. (A-C) Intravascular crawling of Gr-1 labeled neutrophils in venules of the cremaster muscle during superfusion with MIP-2 (5 nM). Displayed are the percentage of adherent cells that crawled (A), mean crawling velocity of adherent cells (B), and mean distance crawled by adherent cells (C) in WT (n = 5) and P-Rex 1^−/− (n = 5) and corresponding experiments after injection of a blocking anti-Mac-1 antibody (n = 3 each) mice ± SEM. (D-F) Isolated P-Rex1^−/− and WT neutrophils were stimulated with MIP-2 and analyzed using a parallel plate flow chamber crawling assay coated with mouse serum. (D) Presented is the mean percentage of cells that remained adherent after applying flow of WT and P-Rex1^−/− neutrophils (10 dyn/cm², 2 minutes) ± SEM (n = 3). (E) Mean crawling velocity before (left), during (middle) and after (right) applying flow (2 dyn/cm²) ± SEM, (F) mean eucledian (left) and accumulated (right) crawled distance ± SEM of WT, P-Rex1^−/− neutrophils and WT cells pretreated with WT or DN peptides for Rac1 or Rac2. (G) Mac-1 activation in HL-60 transfected with either scrambled or P-Rex1 short hairpin RNA after stimulation with IL-8 (n = 3). Bars are mean fluorescence intensity ± SEM as determined by FACS analysis using an antibody specific for the activated conformation of Mac-1. (H) Mac-1 activation after crosslinking of LFA-1 in HL-60 cells (n = 3). Displayed is mean fluorescence intensity ± SEM. (I) Adherent cells in the postcapillary venules of the cremaster muscle in WT and P-Rex1^−/− mice with and without MIP-2 and with and without injection of a blocking anti-Mac-1 antibody. Displayed are adherent cells per mm² ± SEM. (J) Corresponding analysis of transmigrated cells as cells per 1.5 × 10⁴ μm² ± SEM. (K) Mac-1 expression of blood neutrophils with and without MIP-2 stimulation as measured by flow cytometry. Presented as mean fluorescence intensity ± SEM. ^#P < .05.