HUVECs expressing high levels of ICAM-1 support PMN transcellular TEM. (A) Fluorescent micrographs of live HUVEC monolayers treated with media containing TNF-α for 4 hours or 24 hours or media alone (0 hours), as indicated, and then stained with the nonfunction-blocking ICAM-1 mAb CL23.4 conjugated with Alexa 488. The images were captured using identical settings and were processed identically as detailed in “Materials and methods” to create the images shown. Bar, 20 μm, × 40 objective, 0.75 NA. (B) The surface expression of ICAM-1 on 4-hour (purple line) or 24-hour (red line) TNF-α-activated HUVECs was assessed by indirect immunofluorescence staining and flow cytometry as previously described.³⁷  Dashed line indicates nonbinding mAb; solid black line, media alone (0 hours after TNF-α treatment). (C) Confluent primary HUVECs or ICAM-1GFP iHUVECs were treated with TNF-α for 4 hours or 24 hours, as indicated. Endothelial cell monolayers were stained with Alexa 568-conjugated anti-VE-cadherin mAb (Alexa568/hec-1) to identify cell-cell lateral junctions.¹³  The aspect ratio (length to width) was determined by measuring the maximal length and width of the cell outlined by VE-cadherin staining. ^*P ≤ .05. (D) PMNs (5 × 10⁵/mL) in flow buffer (DPBS plus 0.1% HSA) were drawn across control, 4- or 24-hour TNF-α-activated HUVEC monolayers that had been stained with fluorescently labeled VE-cadherin mAb as described in “Materials and methods.” PMN accumulation and location of TEM were determined as follows: sequential DIC and corresponding VE-cadherin red fluorescence images were overlaid in register to create a time-lapse movie using MetaMorph software. The VE-cadherin fluorescence identified the endothelial cell-cell junctions. Neutrophils that arrested or transmigrated at locations greater than 5 μm from cell-cell junctions, as identified by VE-cadherin staining, were considered nonjunctional events. n = 6 from 3 independent experiments. ^*P < .05. The black and gray error bars correspond to paracellular and transcellular route, respectively. All data are mean ± SD. (E) Sequential images recorded by live-cell imaging under high power (× 40 objective), depict a PMN (arrow) that arrested and then transmigrated at a nonjunctional site on 24-hour TNF-α-activated HUVECs. No change in VE-cadherin staining was observed during TEM of this PMN. Bar, 10 μm.