Vav3 is a downstream target of β₂-integrin–dependent macrophage adhesion. (A) Adherence of TNFα-stimulated, CMFDA-loaded WT and Vav3^−/− Mφ plated onto β₁-integrin (fibronectin)–, β₂-integrin (ICAM-1)–, or β₃-integrin (vitronectin)–specific ligands or on 0.1% BSA as control is expressed as percentage of adherent Mφ related to input cells measured after 15 minutes of incubation. Representative results for 3 independent experiments are shown as mean ± SD (n = 3). P assessed by Student t test. (B) Binding of soluble ICAM-1/Fc-FITC by unstimulated (resting) or fMLP-, LTB4- and TNFα-stimulated WT, Vav3^−/− and CD18^−/− Mφ. Results are given in RFU, representing the ratio between the mean green fluorescence intensities of Mφ incubated with ICAM-1/Fc and Mφ incubated with human IgG for each stimulation. Bars indicate means ± SD of triplicate measurements and are representative for 3 different experiments. **P < .005; ***P < .001 by Student t test. (C) Confocal microscopy of WT Mφ plated for 15 minutes on ICAM-1 (top panel) revealed β₂ integrins (CD18) (red) clustering at focal adhesion sites (open and filled arrows). Vav3 (green) colocalized with CD18 in more than 60% of the plated Mφ within the focal adhesion contacts (filled arrows). Colocalization is indicated by the yellow staining. No colocalization occurred between Vav3 and CD29 upon plating Mφ onto fibronectin (bottom panel). (D) Vav3 phosphorylation detected by Vav3 immunoprecipitation and subsequent blotting against anti-phosphotyrosine antibody (pY) induced 10 minutes or 20 minutes after plating Mφ on ICAM-1–coated plates. Total Vav3 levels served as loading controls, Vav3^−/− Mφ served as technical controls. Data are representative of at least 3 independent experiments. (E) Injection of β₂-integrin (CD18)^−/− Vav3 competent Mφ into wound margins of Vav3^−/− mice does not rescue the wound healing defect. WT or CD18^−/− bone marrow–derived Mφ were injected at 4 sites into wound margins of Vav3^−/− mice. Statistical analysis of 16 wounds of each studied time point derived from WT mice or from Vav3^−/− mice after wounding and injection of viable WT Mφ (WT Mφ) or CD18^−/− Mφ (CD18^−/− Mφ⁺). Wound areas are expressed as percentages of the initial (day 0) wound size. Results represented as scatter plots. Bars indicate medians of each cohort (n = 4). *P < .05 by Mann-Whitney test.