Figure 1.
Figure 1. Endothelial tetraspanin proteins relocalize to the contact site with adherent leukocytes and associate with ICAM-1 and VCAM-1. (A) T lymphoblasts were adhered to TNF-α–activated HUVEC monolayers, fixed, and double-stained for CD9 and VCAM-1 or ICAM-1. Maximum projections of the relevant sections from the confocal stacks and the merge of both channels are shown. Asterisks in differential interference contrast (DIC) images highlight the T lymphoblasts around which the docking structures shown in the 3-dimensional (3D) reconstructions are formed. Scale bars equal 10 μm. (B) T lymphoblasts were adhered to TNF-α–activated HUVEC monolayers, fixed, and stained with antitetraspanin mAbs VJ1/20 (anti-CD9), I.33.2.2 (anti-CD81), and LIA1/1 (anti-CD151). Confocal stacks were obtained and representative apical horizontal and vertical sections together with the corresponding DIC images are shown. Arrows point to the positions of the adhered lymphoblasts. Arrowheads and lines point to the position of the adhered lymphoblasts shown in the vertical sections. Scale bar equals 10 μm. (C) Human PBLs, neutrophils, or monocytes were perfused at physiologic flow rate (1.8 dyn/cm2), fixed, and stained with anti-CD9 VJ1/20 mAb. Confocal stacks were obtained and maximum projections of the whole series or a representative section together with the corresponding DIC images are shown. Arrows point to the position of the adhered leukocytes. Scale bar equals 20 μm. (D) Analysis of the localization of endogenous endothelial CD9 or CD151, compared with ICAM-1, at apical and ventral contact sites with transmigrating lymphocytes. Human T lymphoblasts were allowed to transmigrate through TNF-α–activated HUVECs, fixed, and double-stained with antitetraspanin mAbs and biotin-conjugated anti–ICAM-1. Confocal stacks were obtained and representative sections at apical or ventral positions of the same field together with the corresponding DIC image are displayed. White arrows and asterisks mark apically adhered lymphocytes, and gray arrows and black asterisks mark those lymphocytes that have transmigrated. Arrowheads and lines point to the position of the adhered or transmigrated lymphoblast shown in the vertical sections. Scale bar equals 10 μm. (E) ICAM-1 and VCAM-1 are associated with tetraspanins in TNF-α–activated HUVECs. Cell lysates were obtained in 1% Brij96 and immunoprecipitated with the different mAbs specific for endothelial adhesion molecules or tetraspanins. After washing, immunoprecipitates were resolved in sodium dodecyl sulfate–polyacrylamide gel electrophosphoresis (SDS-PAGE) gels and revealed by Western blot for VCAM-1 (P8B1), ICAM-1 (HU5/3), CD151 (8C3) or CD9 (VJ1/20).

Endothelial tetraspanin proteins relocalize to the contact site with adherent leukocytes and associate with ICAM-1 and VCAM-1. (A) T lymphoblasts were adhered to TNF-α–activated HUVEC monolayers, fixed, and double-stained for CD9 and VCAM-1 or ICAM-1. Maximum projections of the relevant sections from the confocal stacks and the merge of both channels are shown. Asterisks in differential interference contrast (DIC) images highlight the T lymphoblasts around which the docking structures shown in the 3-dimensional (3D) reconstructions are formed. Scale bars equal 10 μm. (B) T lymphoblasts were adhered to TNF-α–activated HUVEC monolayers, fixed, and stained with antitetraspanin mAbs VJ1/20 (anti-CD9), I.33.2.2 (anti-CD81), and LIA1/1 (anti-CD151). Confocal stacks were obtained and representative apical horizontal and vertical sections together with the corresponding DIC images are shown. Arrows point to the positions of the adhered lymphoblasts. Arrowheads and lines point to the position of the adhered lymphoblasts shown in the vertical sections. Scale bar equals 10 μm. (C) Human PBLs, neutrophils, or monocytes were perfused at physiologic flow rate (1.8 dyn/cm2), fixed, and stained with anti-CD9 VJ1/20 mAb. Confocal stacks were obtained and maximum projections of the whole series or a representative section together with the corresponding DIC images are shown. Arrows point to the position of the adhered leukocytes. Scale bar equals 20 μm. (D) Analysis of the localization of endogenous endothelial CD9 or CD151, compared with ICAM-1, at apical and ventral contact sites with transmigrating lymphocytes. Human T lymphoblasts were allowed to transmigrate through TNF-α–activated HUVECs, fixed, and double-stained with antitetraspanin mAbs and biotin-conjugated anti–ICAM-1. Confocal stacks were obtained and representative sections at apical or ventral positions of the same field together with the corresponding DIC image are displayed. White arrows and asterisks mark apically adhered lymphocytes, and gray arrows and black asterisks mark those lymphocytes that have transmigrated. Arrowheads and lines point to the position of the adhered or transmigrated lymphoblast shown in the vertical sections. Scale bar equals 10 μm. (E) ICAM-1 and VCAM-1 are associated with tetraspanins in TNF-α–activated HUVECs. Cell lysates were obtained in 1% Brij96 and immunoprecipitated with the different mAbs specific for endothelial adhesion molecules or tetraspanins. After washing, immunoprecipitates were resolved in sodium dodecyl sulfate–polyacrylamide gel electrophosphoresis (SDS-PAGE) gels and revealed by Western blot for VCAM-1 (P8B1), ICAM-1 (HU5/3), CD151 (8C3) or CD9 (VJ1/20).

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