Endothelial transmigration hotspots limit vascular leakage through heterogeneous expression of ICAM1

Upon inflammation, leukocytes leave the circulation by crossing the endothelial monolayer at specific transmigration ‘hotspot’ regions. Although these regions support leukocyte transmigration, their functionality is not clear. We found that endothelial hotspots function to limit vascular leakage during transmigration events. Using the photo-convertible probe mEos4b, we traced back and identified original endothelial transmigration hotspots. Using this method, we show that the heterogeneous distribution of ICAM-1 determines the location of the transmigration hotspot. Interestingly, loss of ICAM-1 heterogeneity either by CRISPR/Cas9-induced knockout of ICAM-1 or equalizing the distribution of ICAM-1 in all endothelial cells results in loss of TEM hotspots but not necessarily in reduced TEM events. Functionally, loss of endothelial hotspots results in increased vascular leakage during TEM. Mechanistically, we demonstrate that the 3 extracellular Ig-like domains of ICAM-1 are crucial for hotspot recognition. However, the intracellular tail of ICAM-1 and the 4th Ig-like dimerization domain are not involved, indicating that intracellular signalling or ICAM-1 dimerization is not required for hotspot recognition. Together, we discovered that hotspots function to limit vascular leakage during inflammation-induced extravasation. * BOEC : Blood Outgrowth Endothelial Cells BSA : Bovine Serum Albumin DMEM : Dulbecco’s Modified Eagle Medium EGM : Endothelial Growth Medium FN : Fibronectin gRNA : Guide RNA HUVEC : Human Umbilical Vein Endothelial Cells ICAM-1/2 : Intercellular Adhesion Molecule Ig : Immunoglobulin IFN : Interferon IL : Interleukin LFA-1 : Lymphocyte-Associated Antigen 1 LPS : Lipopolysaccharide Mac-1 : Macrophage-1 Antigen PBS : Phosphate Buffering Solution P/S : Penicillin/Streptomycin SDM : Site-Directed Mutagenesis TBST : Tris-buffered saline with Tween 20 TEM : Transendothelial Migration TNF : Tumor Necrosis Factor VCAM-1 : Vascular Cell Adhesion Molecule 1 VWF : von Willebrand Factor