Supplementary Material for: ICAM-3 Activation Modulates Cell-Cell Contacts of Human Bone Marrow Endothelial Cells

The Ig-like cell adhesion molecule ICAM-3 is mainly expressed on human leukocytes and is involved in cell-cell interactions. Its expression on endothelium is observed during disorders such as Crohn’s disease and in solid tumors. We found low but detectable expression of ICAM-3 on VE-cadherin-expressing cells from primary human bone marrow aspirates, i.e. endothelial cells, and on primary human endothelial cells from cord blood. Also, immortalized human umbilical cord endothelial cells and human bone marrow endothelial cells showed ICAM-3 expression. However, its function on human endothelium is not known. Surprisingly, activation of endothelial ICAM-3 by crosslinking with specific antibodies resulted in a drop in the electrical resistance of bone marrow endothelial monolayers. In line with this, immunocytochemical analysis showed a loss of endothelial cell-cell contacts after ICAM-3 crosslinking in HBMEC. Detailed biochemical analysis showed an association of moesin and in a later stage ezrin with ICAM-3 upon crosslinking in HBMEC. Moreover, ICAM-3 crosslinking induced the production of reactive oxygen species (ROS), which are known to be involved in the control of endothelial cell-cell contacts. In conclusion, we showed that ICAM-3 is expressed on human bone marrow endothelial cells and controls endothelial integrity via ROS-dependent signaling.