Figure 4
Figure 4. Role of endoglin in leukocyte transmigration. (A-B) Transmigration through endoglin coated membranes. (A) Transwell experiments were performed using 0.5-μm pore membranes previously coated with 1% BSA, 5 μg/mL soluble endoglin or 5 μg/mL soluble endoglin plus 175 ng/mL CXCL12, as indicated. Nalm6 cells were seeded on top of the transwell and 100 ng/mL CXCL12 was added to lower chambers. After incubation for 2 hours at 37°C, transmigrated cells in the bottom chamber were examined by inverse light microscopy (bottom row; magnification ×200) and cells that remained bound to the transwell were stained with propidium iodide (red) and visualized by confocal microscopy (top row; magnification, ×100). (B) Quantification of cells bound to the transwell (red bars) and cells transmigrated (gray bars) was carried out by measuring the surface area of the cells using ImageJ Version 1.46 software. Percentages of the cell signal respect to the total area are indicated. (C-D) Lymphocyte adhesion to HUVECs treated with TNF-α. Confluent monolayers of HUVECs were activated or not with TNF-α for 1 day. Then, culture medium was removed and HUVECs were tested for the adhesion of CFSE-labeled B lymphocytes in the presence CXCL12, soluble endoglin (Eng) or anti–E-selectin mAb (α-Sel), as indicated. (C) After incubation for 1 hour at 37°C, lymphocytes (green) bound to HUVECs were visualized by confocal microscopy (magnification, ×100). (D) Cells in plates were lysed and quantification of bound lymphocytes in the absence (white bars) or in the presence (gray bars) of TNF-α was carried out using a fluorescent analyzer. (E) Lymphocyte transmigration through HUVECs. Confluent monolayers of HUVECs were tested for the migration rate of B lymphocytes in the presence or absence of CXCL12 at the bottom of chambers. Soluble endoglin (Sol-Eng) or anti–E-selectin mAb (α-selectin) were added, as indicated. After incubation for 2 hours at 37°C, transmigrated cells were counted by flow cytometry. (F-G) Immunofluorescence of HUVECs treated with TNF-α. Monolayers of HUVECs were treated or not with TNF-α and incubated with P4A4 mAb (anti-endoglin), followed by the secondary antibody Alexa Fluor 488 anti–mouse IgG. (F) Samples were analyzed using fluorescence confocal microscopy (SP2, Leica). Representative photographs are shown (top 2 rows). The lower row shows the intensity of endoglin staining according to the color scale. (0-200) A diagram of an individual cell (dashed line) is drawn over the pictures. (G) The fluorescence intensity of the whole cell and the cell-cell contact sites was measured with LAS-AF Lite Version 2.4.1 software (Leica). Each value represents the mean average of 60 measurements. The statistical significance of treated versus untreated samples is indicated (**P < .005; *P < .01).

Role of endoglin in leukocyte transmigration. (A-B) Transmigration through endoglin coated membranes. (A) Transwell experiments were performed using 0.5-μm pore membranes previously coated with 1% BSA, 5 μg/mL soluble endoglin or 5 μg/mL soluble endoglin plus 175 ng/mL CXCL12, as indicated. Nalm6 cells were seeded on top of the transwell and 100 ng/mL CXCL12 was added to lower chambers. After incubation for 2 hours at 37°C, transmigrated cells in the bottom chamber were examined by inverse light microscopy (bottom row; magnification ×200) and cells that remained bound to the transwell were stained with propidium iodide (red) and visualized by confocal microscopy (top row; magnification, ×100). (B) Quantification of cells bound to the transwell (red bars) and cells transmigrated (gray bars) was carried out by measuring the surface area of the cells using ImageJ Version 1.46 software. Percentages of the cell signal respect to the total area are indicated. (C-D) Lymphocyte adhesion to HUVECs treated with TNF-α. Confluent monolayers of HUVECs were activated or not with TNF-α for 1 day. Then, culture medium was removed and HUVECs were tested for the adhesion of CFSE-labeled B lymphocytes in the presence CXCL12, soluble endoglin (Eng) or anti–E-selectin mAb (α-Sel), as indicated. (C) After incubation for 1 hour at 37°C, lymphocytes (green) bound to HUVECs were visualized by confocal microscopy (magnification, ×100). (D) Cells in plates were lysed and quantification of bound lymphocytes in the absence (white bars) or in the presence (gray bars) of TNF-α was carried out using a fluorescent analyzer. (E) Lymphocyte transmigration through HUVECs. Confluent monolayers of HUVECs were tested for the migration rate of B lymphocytes in the presence or absence of CXCL12 at the bottom of chambers. Soluble endoglin (Sol-Eng) or anti–E-selectin mAb (α-selectin) were added, as indicated. After incubation for 2 hours at 37°C, transmigrated cells were counted by flow cytometry. (F-G) Immunofluorescence of HUVECs treated with TNF-α. Monolayers of HUVECs were treated or not with TNF-α and incubated with P4A4 mAb (anti-endoglin), followed by the secondary antibody Alexa Fluor 488 anti–mouse IgG. (F) Samples were analyzed using fluorescence confocal microscopy (SP2, Leica). Representative photographs are shown (top 2 rows). The lower row shows the intensity of endoglin staining according to the color scale. (0-200) A diagram of an individual cell (dashed line) is drawn over the pictures. (G) The fluorescence intensity of the whole cell and the cell-cell contact sites was measured with LAS-AF Lite Version 2.4.1 software (Leica). Each value represents the mean average of 60 measurements. The statistical significance of treated versus untreated samples is indicated (**P < .005; *P < .01).

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