Biology subjects

Kempers, L.

Publications and source records attributed to Kempers, L..

3 recordsLinked to original sources

bioRxiv2024

Leukocytes use endothelial membrane tunnels to extravasate the vasculature

Upon inflammation, leukocytes extravasate through endothelial cells. When they extravasate in a paracellular manner, it is generally accepted that neighbouring endothelial cells physically disconnect to open cell-cell junctions, allowing leukocytes to cross. When carefully examining endothelial junctions, we found a partial membrane overlap of endothelial cells beyond VE-cadherin distribution. These overlaps are regulated by actin polymerization and, although marked by, do not require PECAM-1, nor VE-cadherin. Neutrophils prefer wider membrane overlaps as exit sites. Detailed 3D analysis of endothelial membrane dynamics during paracellular neutrophil transmigration in real-time, at high spatiotemporal resolution using resonant confocal and lattice light-sheet imaging, revealed that overlapping endothelial membranes form a tunnel during neutrophil transmigration. These tunnels are formed by the neutrophil lifting the membrane of the upper endothelial cell while indenting and crawling over the membrane of the underlying endothelial cell. Our work shows that endothelial cells do not simply retract upon passage of neutrophils but provide membrane tunnels, allowing neutrophils to extravasate. This discovery defines the 3D multicellular architecture in which the paracellular transmigration of neutrophils occurs.

cell biology↗

bioRxiv2022

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.

immunology↗

bioRxiv2021

Transendothelial migration induces differential migration dynamics of leukocytes in tissue matrix

Leukocyte extravasation into inflamed tissue is a complex process that is difficult to capture as a whole in vitro. We employed a blood-vessel-on-a-chip model in which endothelial cells were cultured in a tube-like lumen in a collagen-1 matrix. The vessels are leak-tight, creating a barrier for molecules and leukocytes. Addition of inflammatory cytokine TNF- caused vasoconstriction, actin remodelling and upregulation of ICAM-1. Introducing leukocytes into the vessels allowed real-time visualisation of all different steps of the leukocyte transmigration cascade including migration into the extracellular matrix. Individual cell tracking over time distinguished striking differences in migratory behaviour between T-cells and neutrophils. Neutrophils cross the endothelial layer more efficiently than T-cells, but upon entering the matrix, neutrophils display high speed but low persistence, whereas T-cells migrate with low speed and rather linear migration. In conclusion, 3D imaging in real-time of leukocyte extravasation in a vessel-on-a-chip enables detailed qualitative and quantitative analysis of different stages of the full leukocyte extravasation process in a single assay. SummaryA functional hydrogel-based blood-vessel-on-a-chip model is used to study the complete leukocyte transendothelial migration process in real time. T-lymphocytes and neutrophils exhibit distinct migration dynamics in the extravascular matrix after transendothelial migration, which can be altered using a chemotactic gradient.

cell biology↗