Defective release of active TGF-β₁ is due to impaired adhesion resulting in defective phagocytosis of apoptotic PMN by macrophages in Vav3^−/− mice. (A) In vitro adhesion and phagocytosis of WT and Vav3^−/− Mφ cocultured with CMRA-labeled Vav1^−/−/Vav3^−/− or WT apoptotic PMN for 15 minutes (adhesion; left panel) or 45 minutes (phagocytosis; right panel) assessed by flow cytometry as CMRA⁺F4/80⁺ conjugates. Results are expressed as percentages of PMN-binding Mφ of the total Mφ count (PMN-binding Mφ × 100/total number of Mφ). Each symbol indicates the median of a triplicate analysis. *P < .05; **P < .005. (B) Active TGF-β₁ concentrations measured by ELISA in supernatants of unstimulated WT and Vav3^−/− Mφ and in cocultures between Mφ and PMN of the indicated genotypes. Data given as mean ± SD (n = 4). **P < .005. (C) Active TGF-β₁–producing Mφ (yellow) identified by immunostaining of cryosections from 3- and 5-day-old Vav3^−/− and WT wounds for TGF-β₁ (red) and F4/80 (green). Cell nuclei are counterstained with DAPI (blue; original magnification ×20, scale bar indicates 150 μm; he, hyperproliferative epidermis; we, wound edge; and gt, granulation tissue). (D) Statistical analysis of 16 wounds derived from WT mice and from Vav3^−/− mice after wounding and injection of viable WT Mφ (WT Mφ⁺) or of Vav3^−/− Mφ (Vav3^−/− 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. (E) Quantitative evaluation of active TGF-β₁ measured by specific ELISA in lysates of 5- and 7-day-old wounds of WT mice and Vav3^−/− mice after wounding and injection of viable WT Mφ (WT Mφ+) or Vav3^−/− Mφ (Vav3^−/− Mφ+) into wound margins. Results representative of 2 independent experiments are expressed as mean plus or minus SD (n = 4). *P < .05 by Student t test. (F) Lethally irradiated WT mice reconstituted with bone marrow from Vav3^−/− mice reveal a significant delay in wound healing. Statistical analysis of 16 wounds per studied time point derived from lethally irradiated WT mice reconstituted with bone marrow from either WT (■) or Vav3^−/− (□) mice were subjected to image analysis. Wound areas are expressed as percentages of the initial (day 0) wound size. Results given as mean ± SD (n = 4) reflect 1 of 2 independent experiments. *P < .05 by Mann-Whitney test. (G) Western blot analysis of expression levels of αSMA, TGFβ-RII and PECAM-1 equilibrated to total actin and vimentin levels in wound margins of WT and Vav3^−/− bone marrow chimeric mice at days 5 and 7 after wounding (cWT, WT chimera with WT bone marrow; cVav3^−/−, WT chimera with Vav3^−/− bone marrow).