Inside-out regulation of ICAM-1 dynamics in TNF-alpha-activated endothelium

Abstract

Background: During transendothelial migration, leukocytes use adhesion molecules, such as ICAM-1, to adhere to the endothelium. ICAM-1 is a dynamic molecule that is localized in the apical membrane of the endothelium and clusters upon binding to leukocytes. However, not much is known about the regulation of ICAM-1 clustering and whether membrane dynamics are linked to the ability of ICAM-1 to cluster and bind leukocyte integrins. Therefore, we studied the dynamics of endothelial ICAM-1 under non-clustered and clustered conditions.

Principal findings: Detailed scanning electron and fluorescent microscopy showed that the apical surface of endothelial cells constitutively forms small filopodia-like protrusions that are positive for ICAM-1 and freely move within the lateral plane of the membrane. Clustering of ICAM-1, using anti-ICAM-1 antibody-coated beads, efficiently and rapidly recruits ICAM-1. Using fluorescence recovery after photo-bleaching (FRAP), we found that clustering increased the immobile fraction of ICAM-1, compared to non-clustered ICAM-1. This shift required the intracellular portion of ICAM-1. Moreover, biochemical assays showed that ICAM-1 clustering recruited beta-actin and filamin. Cytochalasin B, which interferes with actin polymerization, delayed the clustering of ICAM-1. In addition, we could show that cytochalasin B decreased the immobile fraction of clustered ICAM-1-GFP, but had no effect on non-clustered ICAM-1. Also, the motor protein myosin-II is recruited to ICAM-1 adhesion sites and its inhibition increased the immobile fraction of both non-clustered and clustered ICAM-1. Finally, blocking Rac1 activation, the formation of lipid rafts, myosin-II activity or actin polymerization, but not Src, reduced the adhesive function of ICAM-1, tested under physiological flow conditions.

Conclusions: Together, these findings indicate that ICAM-1 clustering is regulated in an inside-out fashion through the actin cytoskeleton. Overall, these data indicate that signaling events within the endothelium are required for efficient ICAM-1-mediated leukocyte adhesion.

Figure 1. ICAM-1 clusters to leukocyte adhesion sites.

(A) Left panel shows localization of endogenous ICAM-1 in green on endothelial filopodia (Arrowheads). Middle panel shows localization of ICAM-1-GFP, expressed in HeLa cells on filopodia. Right panel shows scanning electron microscopy (SEM) recording of TNF-α-pretreated HUVECs visualising small filopodia protruding out of the apical plane of the membrane (arrowheads). Bars, 10 µm (B) Cos7 cells expressing GFP (left panel) or ICAM-1-GFP (right panel) were cultured on glass cover slips and analyzed by SEM. ICAM-1-GFP but not GFP only-expressing Cos7 cells showed multiple filopodia (arrowheads). Bar, 10 µm. (C) TNF-α-pretreated HUVECs were cultured on glass cover slips and calcein-red labelled differentiated HL60 cells were allowed to adhere for 30 minutes. Arrowheads show recruitment of ICAM-1 to adherent HL60 cells. Images show ICAM-1 in green and HL60 cells in red. Right panel shows ICAM-1-GFP-transfected HeLa cells, cultured on glass cover slips and calcein-red labelled differentiated HL60 cells adhered for 30 minutes. Bar, 10 µm. (D) ICAM-1-GFP-transfected Cos7 cells were cultured on glass covers and αICAM-1-antibody coated beads (size 3 µm) adhered for 30 minutes. Arrowheads show recruitment of ICAM-1-GFP to adhered beads. Images show ICAM-1-GFP in green, F-actin in red, merge in yellow and the beads in white. Note that the beads specifically adhered to ICAM-1-GFP expressing cells. The asterisk shows non-transfected cell with no bead adhesion. Bar, 20 µm. (E) ICAM-1-GFP (green) is expressed in HeLa cells and recruited (arrowheads) to αICAM-1-antibody coated beads (size 10 µm; asterisks). Quantification of ICAM-1 recruitment on the right shows that ICAM-1-GFP is recruited to 25% of the beads after 36 minutes. Data are mean ± SEM from four experiments.

Figure 2. Lateral mobility of ICAM-1 depends on its C-terminal domain.

(A) ICAM-1-GFP is expressed in HeLa cells and FRAP was measured. Box in the upper panel depicts the FRAP region (square) and the recovery in time after every 10 seconds. Image at the right shows recovery after 200 seconds. Middle panel shows ICAM-1-GFP FRAP that is clustered around an adherent bead, coated with ICAM-1 antibodies. Lower image shows the merge of the green channel and DIC, in which the bead is visible. Bars, 10 µm. (B) Quantification of the FRAP data show decreased ICAM-1-GFP mobility following clustering. Graph on the left shows ICAM-1-GFP FRAP in time and graph on the right shows the difference in ICAM-1 mobility at t = 300 seconds. ICAM-1 was clustered using αICAM-1 antibody-coated beads. ICAM-1 FRAP was significantly reduced upon clustering. Experiment is carried out 6 times. Data are mean ± SEM. *p<0.01. (C) TNF-α-pretreated HUVECs were cultured on FN-coated surfaces and anti-ICAM-1-coated magnetic beads were allowed to adhere to the endothelium for 30 minutes, resulting in clustering of ICAM-1, after which the cells were lysed. As a control, the beads were added to endothelial cell lysates. Lysate conditions prevent ICAM-1 from clustering and these samples are therefore marked as Non-clustered. Next, using a magnetic pen, the beads were isolated from the cell lysates, washed and analyzed using Western blotting. The images on the left show that the magnetic beads efficiently precipitated ICAM-1 and that ICAM-1 clustering resulted in the recruitment of actin and filamin A and B. Images on the right show the expression of indicated proteins in the cell lysates.

Figure 3. Requirement of the intracellular domain of ICAM-1 for clustering.

(A) ICAM-1-GFP full length or a construct lacking the intracellular tail (ICAM-1-ΔC) were transfected in HeLa cells and cultured on glass covers. Full-length ICAM-1 localizes to filopodia whereas the ΔC-mutant shows a more dispersed distribution. Images show ICAM-1 staining in green, F-actin in red and merge in yellow. Image on the right is a magnification of the merge. Bars, 20 µm. (B) ICAM-1-GFP full length or ΔC was transfected in Cos7 cells that lack basal filopodia and images were taken using a scanning electron microscope. Zoom shows filopodia in full-length or ICAM-1 ΔC expressing Cos7 cells, Bar, 1 µm. (C) Quantification of filopodia in Cos7 cells shows that ICAM-1 full length induces filopodia with an average length of 750nm. The ΔC-mutant shows smaller and fewer filopodia. Bar graph shows number of filopodia per Cos7 cell. Experiment is done three times. Data are mean ± SEM. *p<0.01. **p<0.001. (D) Full length and ΔC ICAM-1-GFP are expressed in HeLa cells. Left panel shows FRAP in time and right panel shows FRAP quantification after 5 minutes of bleaching. ICAM-1-ΔC recovers significantly faster than full length ICAM-1. Experiment is done four times. Data are mean ± SEM. *p<0.05. (E) Full length and ΔC ICAM-1-GFP are expressed in HeLa cells and ICAM-1 clustering was induced through αICAM-1 antibody-coated beads. Left panel shows FRAP in time and right panel shows FRAP quantification after 5 minutes of bleaching. Clustered ICAM-1-ΔC recovers significantly faster than full length. Experiment is done four times. Data are mean ± SEM. *p<0.05.

Figure 4. Actin polymerization is required for the shift of ICAM-1 to the immobile fraction.

(A) HUVECs were treated with Cytochalasin B (CytoB; 5 µg/mL) for 10 minutes to block actin polymerization, fixed and analyzed by fluorescent microscopy. CytoB prevents ICAM-1 localization to filopodia. Images show ICAM-1 staining in green and F-actin in red. Merge shows co-localization of ICAM-1 and F-actin. Bar, 20 µm. (B) ICAM-1-GFP is expressed in HeLa cells and treated or not with CytoB. ICAM-1 is clustered as described in B. Left panel shows no difference in FRAP of non-clustered ICAM-1 upon CytoB treatment. Panel at the right shows increased ICAM-1 FRAP when actin polymerization was blocked. Experiment is done three times. Data are mean ± SEM. *p<0.05. (C) Quantification of clustered and non-clustered ICAM-1 shows that ICAM-1-GFP recruitment upon clustering is delayed upon CytoB treatment but not in non-clustered conditions. Experiment is repeated 3 times and data are mean ± SEM. *p<0.05. (D) ICAM-1-GFP is expressed in HeLa cells and recruited to αICAM-1-antibody coated beads as described under 1E. Quantification graph shows that ICAM-1-GFP recruitment is delayed when actin polymerization is blocked. Experiment is repeated 3 times and data are mean ± SEM. *p<0.05. (E) TNF-α-pretreated HUVECs were cultured on FN-coated surfaces and prior to anti-ICAM-1-coated magnetic beads-induced clustering, cells were pretreated with CytoB or jasplakinolide (10 µM). Next, beads were allowed to adhere to the endothelium for 30 minutes, and samples were processed as described in the legend of figure 2C. Panels on the left show magnetic beads pull down. ICAM-1 was precipitated equally under all conditions. Clustering of ICAM-1 induced the link to actin, which was prevented by CytoB pre-treatment. Stabilizing actin filaments by jasplakinolide promoted the link of ICAM-1 with actin upon clustering. Panels on the right show the expression of indicated proteins in the cell lysates.

Figure 5. ICAM-1 requires filamin and its intracellular tail for the actin link and its motility.

(A) TNF-α-pretreated HUVECs were cultured on FN-coated surfaces and filamin expression was reduced using siRNA, according to Kanters et al. . Next, anti-ICAM-1-coated magnetic beads were allowed to adhere to the endothelium for 30 minutes, and samples were processed as described in the legend of figure 2C. Panels on the left show magnetic beads pull down. ICAM-1 was precipitated equally in both conditions. Clustering of ICAM-1 induced the link to actin, which was prevented by silencing Filamin A. Panels on the right show the expression of indicated proteins in the cell lysates. (B) Full length (FL) and ΔC ICAM-1-GFP are expressed in M2 and A7 cells. Panel shows FRAP quantification. The data show that the recovery of full length ICAM-1 is faster in the absence of filamin, e.g. in M2 cells. ICAM-1-ΔC-GFP FRAP is independent on the presence of filamin. Experiment is done three times. Data are mean ± SEM. *p<0.05. (C) Cells were transfected as described in B. ICAM-1-Ab coated beads were allowed to adhere for 30 minutes to cluster ICAM-1. FRAP was measured at sites of bead adhesion in A7 (left panel) and M2 cells (right panel). Lower panel shows FRAP quantification. The data show that ICAM-1 requires filamin and its intracellular tail to shift to the immobile fraction upon clustering. Experiment is done four times. Data are mean ± SEM. *p<0.05.

Figure 6. Myosin and lipid raft formation are required for the shift of ICAM-1 to the immobile fraction.

(A) TNF-α-pretreated HUVECs were cultured on glass covers and images show ICAM-1 staining in green using ICAM-1-FITC to avoid cross-talk with the beads, myosin-II in red, merge of ICAM-1 and myosin-II in yellow, F-actin in white and DIC is included to depict the beads (arrowheads). Myosin-II together with ICAM-1 is clustered around the αICAM-1-antibody coated beads that were allowed to adhere for 30 minutes. Bar, 20 µm. Lower panel shows X-Z projection of ICAM-1 in green and myosin-II in red (arrowheads) around the adhered αICAM-1-antibody coated beads (asterisk). White depicts F-actin. Bar, 5 µm. (B) Cells were pretreated with 8 µM Blebbistatin to inhibit myosin-II for 30 minutes (Blebbi). ICAM-1-GFP FRAP was analyzed as described above and showed that Blebbistatin significantly decreased the FRAP of non-clustered and clustered ICAM-1-GFP. Experiment is done three times. Data are mean ± SEM. *p<0.05. (C) Cells were pretreated with 5mM methyl-β-cyclodextrin for 30 minutes (β-CD). ICAM-1-GFP FRAP was analyzed as described above and showed that β-CD did not affect ICAM-1-GFP FRAP of non-clustered ICAM1-GFP but significantly reduced the FRAP of clustered ICAM-1-GFP. Experiment is done three times. Data are mean ± SEM. *p<0.01.

Figure 7. Rac1 activity is required for optimal ICAM-1 dynamics.

(A) Cells were pretreated with 100 µM NSC-23766 (NSC) for 30 minutes. Quantification of ICAM-1 recruitment using αICAM-1 antibody coated beads shows that ICAM-1-GFP recruitment is reduced upon inhibition of Rac1 activity by NSC. Experiment is repeated 3 times and data are mean ± SEM. *p<0.05. (B) ICAM-1-GFP FRAP was analyzed as described in Materials and Methods and showed that NSC reduced FRAP of non-clustered and clustered ICAM-1-GFP. Experiment is done three times. Data are mean ± SEM. *p<0.05.

Figure 8. Src-kinase has no effect on ICAM-1 dynamics.

(A) Analysis of Src, Yes and Fyn-kinase (SYF)-deficient (SYF^-/-) or SYF^-/- with reconstituted Src (SYF^-/-+Src) or control (CTRL) mouse embryonic fibroblast (MEF) cells by western blotting using antibodies against Src, Yes or Fyn. Tubulin was used for protein loading control. (B) Immunofluorescent images show localization of ICAM-1-GFP in filopodia in SYF^-/- or SYF^-/-+Src MEF cells. (C) Graph shows that the expression of Src had only minor effect on the ICAM-1 FRAP in the SYF^-/- cells. (D) αICAM-1 antibody coated beads (size 3 µm) were allowed to adhere for 30 minutes on ICAM-1-GFP transfected SYF^-/- or SYF^-/-+Src cells. Number of beads per ICAM-1-GFP-positive cell was counted. No difference in adhesion of these beads the MEF cells is measured. Experiment was carried out three times.

Figure 9. Quantification of ICAM-1 adhesion.

(A) αICAM-1-antibody coated-beads were allowed to pre-adhere to TNF-α-stimulated endothelium for 5 minutes. Experiment was carried out as described in Materials and Methods. Briefly, low shear was introduced (0.25dyn/cm²) and flow was increased stepwise with 1.25dyn/cm² at the time points, indicated with arrows. The final shear corresponded to 6.25dyn/cm². The αICAM-1-antibody coated beads detached from the endothelium when shear increased. Pre-treatment of the endothelium with cytochalasin B (CytoB) or NSC-23766 (NSC) showed a steeper decline of detached beads upon increased shear flow. Experiment is done three times. Data are mean ± SEM. *p<0.05. (B) Experiment carried out as described under A. Pre-treatment of the endothelial cells with either blebbistatin (Blebbi) or cyclodextrin (β-CD) show a decrease in adhesion of the beads to the endothelium, already after the first increase in shear flow. Experiment is done three times in duplicate. Data are mean ± SEM. *p<0.01.

Figure 10. Schematic overview of ICAM-1 dynamics.

ICAM-1 dynamics require Rac1 and myosin-II activity. Upon clustering, actin polymerization and lipid raft formation play a pivotal role. These signalling modules all affect the adhesive capacity of ICAM-1 under flow conditions.