Thesis Bram van Steen

Summary

Under inflammatory conditions, leukocytes are directed to exit the vasculature and pass through the endothelial barrier. In this thesis, we focus on specific aspects of the multistep transendothelial migration (TEM) process. First, we give a literature overview of the changes in endothelial actin dynamics upon inflammation and during leukocyte TEM that facilitate this process. Next, we performed a proteomics analysis of the ICAM-1 adhesome that is recruited at the docking site of the leukocyte, mapping the entire complex for the first time. We follow up on this screen by demonstrating the importance of CD44 for efficient cytotoxic T-lymphocyte (CTL) extravasation. Another follow-up study demonstrated that CTL form a transmigration synapse that is essential extravasation via the transcellular route. In the second part of this thesis, we generated a new leukocyte TEM assay by adapting blood-vessel-on-a-chip (BVOAC) technology. Current models to study leukocyte extravasation are based on endothelium cultured on stiff surfaces, which means TEM can only be studied until the point where leukocytes cross the endothelium. We demonstrate that BVOAC can be used to study the complete leukocyte TEM process from the vascular lumen into the perivascular tissue matrix with real-time and 3D microscopy. We applied this model to characterize migration dynamics induced by the endothelium in CTL and neutrophils. Finally, we show with our BVOAC model that neutrophils from a patient with a deficiency in the ARPC1B gene are impaired in both traversing the endothelium and migration into the tissue matrix.