Figure 2
Figure 2. Cytohesin-1 (red fluorescence) colocalizes with CD18 (green fluorescence) and mediates both CD18 activation and adhesion. In contrast to unstimulated mature Mo-DCs where cytohesin-1 (detected by mAb 7H2) localizes to the cytoplasm (A top panel), stimulation with 200 ng/mL CCL19 for 10 minutes induces translocation of cytohesin-1 to the plasma membrane (A middle panel) where it colocalizes with beta-2 integrins, clustered by an anti-CD18 antibody (A bottom panel; bar represents 5 μm). All shown fluorescence staining images are confocal images, focused on the medial z-position of the respective cells, that is, the cytoplasm and the plasma membrane (A). The different fluorochromes of secondary antibodies in the costaining of cytohesin-1 and CD18 were acquired sequentially by the use of a confocal laser scanning microscope (A). Flow cytometric analysis shows that CCL19 (200 ng/mL for 10 minutes) strongly increases the expression of the high-affinity state (detected by mAb 327C) of LFA-1 (B). RNAi of cytohesin-1 reduces expression of the activation epitope, compared with control cells (B). In contrast, a constitutive CD18 epitope (detected by mAb MHM23) is not altered after RNAi of cytohesin-1 (B). The empty profiles in the flow cytometry histograms indicate the background cell stainings by isotype controls (B). RNAi of cytohesin-1 strongly reduces static adhesion of Mo-DCs to ICAM-1-Fc compared with control cells (C). Cytohesin-1 siRNAs selectively down-regulate antigen-specific BM-DC adhesion to OT-1 T cells (D). Under flow conditions, RNAi of cytohesin-1 reduces dynamic adhesion of Mo-DCs to human umbilical vein endothelial cells (HUVECs; E) or of BM-DCs to brain endothelioma cells (B end5; F). Overexpression of wild-type cytohesin-1 but not of the GEF-deficient E157K mutant increases static adhesion of GFP-cotransfected Mo-DCs to ICAM-1-Fc (G). In the static adhesion assays, Mo-DCs were stimulated with 50 ng/mL PMA for 60 minutes at 37°C (C,G). Error bars indicate ± SD (C-G). ***P < .001, **P < .01, *P < .05. Each experiment was repeated at least 3 times independently. Each single experiment was performed in duplicate.

Cytohesin-1 (red fluorescence) colocalizes with CD18 (green fluorescence) and mediates both CD18 activation and adhesion. In contrast to unstimulated mature Mo-DCs where cytohesin-1 (detected by mAb 7H2) localizes to the cytoplasm (A top panel), stimulation with 200 ng/mL CCL19 for 10 minutes induces translocation of cytohesin-1 to the plasma membrane (A middle panel) where it colocalizes with beta-2 integrins, clustered by an anti-CD18 antibody (A bottom panel; bar represents 5 μm). All shown fluorescence staining images are confocal images, focused on the medial z-position of the respective cells, that is, the cytoplasm and the plasma membrane (A). The different fluorochromes of secondary antibodies in the costaining of cytohesin-1 and CD18 were acquired sequentially by the use of a confocal laser scanning microscope (A). Flow cytometric analysis shows that CCL19 (200 ng/mL for 10 minutes) strongly increases the expression of the high-affinity state (detected by mAb 327C) of LFA-1 (B). RNAi of cytohesin-1 reduces expression of the activation epitope, compared with control cells (B). In contrast, a constitutive CD18 epitope (detected by mAb MHM23) is not altered after RNAi of cytohesin-1 (B). The empty profiles in the flow cytometry histograms indicate the background cell stainings by isotype controls (B). RNAi of cytohesin-1 strongly reduces static adhesion of Mo-DCs to ICAM-1-Fc compared with control cells (C). Cytohesin-1 siRNAs selectively down-regulate antigen-specific BM-DC adhesion to OT-1 T cells (D). Under flow conditions, RNAi of cytohesin-1 reduces dynamic adhesion of Mo-DCs to human umbilical vein endothelial cells (HUVECs; E) or of BM-DCs to brain endothelioma cells (B end5; F). Overexpression of wild-type cytohesin-1 but not of the GEF-deficient E157K mutant increases static adhesion of GFP-cotransfected Mo-DCs to ICAM-1-Fc (G). In the static adhesion assays, Mo-DCs were stimulated with 50 ng/mL PMA for 60 minutes at 37°C (C,G). Error bars indicate ± SD (C-G). ***P < .001, **P < .01, *P < .05. Each experiment was repeated at least 3 times independently. Each single experiment was performed in duplicate.

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