Abstract
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.
Abbreviations
- 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