Figure 4.
Figure 4. PMNs treated with anti-CD157 or obtained from patients with PNH show reduced transendothelial migration. (A) PMNs from healthy donors were labeled with CFSE, incubated with anti-CD157, anti-CD31, or irrelevant mAb (20 μg/mL), and seeded on TNF-α–activated HUVEC monolayers grown on collagen. After 30 minutes of migration, samples were fixed, washed, stained with anti-CD31 mAb and F(ab′)2-GαMIg-Texas red and evaluated by laser-scanning confocal microscopy (bar represents 30 μm). The images show the position of neutrophils at the end of transmigration assays. No quantitative information must be inferred. Cells were imaged using a × 60 oil immersion objective (1.4 NA). Samples were analyzed by sequential scanning of the xy planes recorded along the z-axis (step size: 1 μm). Series of confocal optical xy images were processed using a 3-dimensional reconstruction program (FluoView 300 software) and visualized as orthogonal views. (B) PMNs were seeded on TNF-α–activated HUVEC monolayers grown on fibronectin-coated transwell filters in the presence of the indicated mAb (20 μg/mL). After 1 hour of incubation at 37°C, the transmigrated cells were counted. Results are expressed as the percentage of transmigrated cells. Data are mean ± SD of 6 experiments performed in triplicate. *P < .01 anti-CD157 mAb and anti-CD31 mAb versus irrelevant IgG. (C) CFSE-labeled PMNs from patients with PNH were seeded on activated HUVECs, incubated, and analyzed as in panel A. Representative experiments from 2 PNH patients are shown. Similar results were observed in all the patients analyzed. (D) Transendothelial migration of PMNs from a representative PNH patient with recurrent bacterial infections analyzed by laser-scanning confocal microscopy, and (E) conventional transmigration assay across TNF-α–activated HUVECs monolayers grown on fibronectin-coated transwell filters. Migration is shown in the presence of a 10-nM fMLP transwell gradient. The difference between transmigration of PMNs from a PNH patient versus PMNs from healthy donor was significant, with P < .001 at any time considered.

PMNs treated with anti-CD157 or obtained from patients with PNH show reduced transendothelial migration. (A) PMNs from healthy donors were labeled with CFSE, incubated with anti-CD157, anti-CD31, or irrelevant mAb (20 μg/mL), and seeded on TNF-α–activated HUVEC monolayers grown on collagen. After 30 minutes of migration, samples were fixed, washed, stained with anti-CD31 mAb and F(ab′)2-GαMIg-Texas red and evaluated by laser-scanning confocal microscopy (bar represents 30 μm). The images show the position of neutrophils at the end of transmigration assays. No quantitative information must be inferred. Cells were imaged using a × 60 oil immersion objective (1.4 NA). Samples were analyzed by sequential scanning of the xy planes recorded along the z-axis (step size: 1 μm). Series of confocal optical xy images were processed using a 3-dimensional reconstruction program (FluoView 300 software) and visualized as orthogonal views. (B) PMNs were seeded on TNF-α–activated HUVEC monolayers grown on fibronectin-coated transwell filters in the presence of the indicated mAb (20 μg/mL). After 1 hour of incubation at 37°C, the transmigrated cells were counted. Results are expressed as the percentage of transmigrated cells. Data are mean ± SD of 6 experiments performed in triplicate. *P < .01 anti-CD157 mAb and anti-CD31 mAb versus irrelevant IgG. (C) CFSE-labeled PMNs from patients with PNH were seeded on activated HUVECs, incubated, and analyzed as in panel A. Representative experiments from 2 PNH patients are shown. Similar results were observed in all the patients analyzed. (D) Transendothelial migration of PMNs from a representative PNH patient with recurrent bacterial infections analyzed by laser-scanning confocal microscopy, and (E) conventional transmigration assay across TNF-α–activated HUVECs monolayers grown on fibronectin-coated transwell filters. Migration is shown in the presence of a 10-nM fMLP transwell gradient. The difference between transmigration of PMNs from a PNH patient versus PMNs from healthy donor was significant, with P < .001 at any time considered.

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