ENO-1 overexpression facilitates recruitment of monocytic cells to the alveolar compartment of LPS-treated mice. BALB/c mice were challenged intratracheally with either saline or LPS (0.5 mg/kg b.w.). After 24 hours, mice received an intravenous injection of PKH26-PCL–labeled (red color) stably transfected U937 cells (5 × 106 in a total volume of 100 μL PBS). Mice were then killed 5 hours after injection. (A) Representative lung tissue sections from saline- (left panel) or LPS- (right panel) treated mice injected with untransfected cells (−), mock-transfected cells (pcDNA3.1), cells overexpressing ENO-1 wild-type (ENO-1WT), or cells overexpressing ENO-1 lacking the plasminogen binding site (ENO-1ΔPLG). Original magnification ×20. Bar size 20 μm. Insert, original magnification 63×/1.32-0.6 oil objective. (B) Relative intensity of red/blue staining presented in panel A. Relative intensity of red/blue staining was determined by counting 4 randomly chosen fields per section using 3 independent sections for each group. □ represents saline-treated animals, while ■ represents LPS-treated mice. **P < .01; ***P < .001 (C) Localization of PKH26-PCL–positive monocytic cells overexpressing ENO-1WT (red color) within the alveolar wall. Sections were immunostained with the endothelial marker von Willebrand factor (VWF; green color; left panel) or with the alveolar epithelial type II cell marker prosurfactant protein-C (pro-SP-C; green color; right panel). EC indicates endothelial cells; and ATII, alveolar epithelial type II cells. Original magnification 63×/1.32-0.6 oil objective. Bar size 5 μm. (D) Fluorescence signal from PKH26-PCL–labeled monocytic cells present in bronchoalveolar lavage fluids obtained from saline- (□) or LPS- (■) treated mice. Mean values ± SD are reported; with n = 10 for each group; ***P < .001. (E) Representative lung tissue sections from LPS-treated mice injected with PKH26-PCL–labeled (red color) untransfected cells (−), mock-transfected cells (pcDNA3.1), cells overexpressing ENO-1 wild-type (ENO-1WT), or cells overexpressing ENO-1 lacking the plasminogen binding site (ENO-1ΔPLG) stained for PLG (green color). Original magnification 40×/1.25-0.75 oil objective. Bar size 10 μm. (F) Cellular localization of ENO-1 and PLG in U937 cells overexpressing ENO-1 recovered from bronchoalveolar lavage fluid of LPS-treated mice. Original magnification 63×/1.32-0.6 oil objective. Bar size 5 μm.
