Migrationsmechanismen von Lymphozyten durch die Blut-Hirn-Schranke, untersucht anhand eines in vitro Modells, das den Blutstrom nachahmt und eine Entzündung simuliert - wie es bei Multipler Sklerose, der häufigsten Autoimmunerkrankung des Zentralnervensystems, der Fall ist.
Zusammenfassung der Projektergebnisse
Infiltration of immune cells across the blood-brain barrier (BBB) is a key step in many neurological diseases, including multiple sclerosis. Drugs like natalizumab, that prevent autoreactive immune cells from entering the brain, are therefore beneficial. In order to better understand molecular mechanisms of leukocyte trafficking at the BBB and to study how to manipulate this process for potential therapeutic applications, we developed an advanced fully-human in vitro BBB model comprising endothelial cell/astrocyte co-culture, and allowing simulation of blood flow conditions in the setting of inflammation. We observed that co-culture with astrocytes helps to maintain endothelial cell integrity, particularly when exposed to cytokine and shear stress, i.a. in an S1P receptor mediated manner. By establishing more read-outs, particularly the classification of luminally-attached cells in relation to endothelial cell boundaries, we found that leukocytes preferentially attach to junctions of adjacent endothelial cells. This phenomenon is likely to depend on CD31 and CD99. In addition, we have adjusted our system for live cell videomicroscopy, which enables us to study dynamics of leukocyte trafficking, i.a. the poorly understood crawling behavior, under flow in real time. Proof of concept experiments confirmed the reliability of our in vitro system, since we successfully reproduced rolling, sticking, crawling, and transmigration in discrete steps as reported from in vivo imaging. Using flow-based approaches, we will also further address the surprising phenomenon of chemokine aggregation on the luminal endothelial surface, which we initially discovered and characterized under static conditions. The most significant surprise was the fact that I could not execute my project as planned since the proposal was based on findings that were not reproducible for a number of reasons. However, this allowed me to design an entirely new line of research to study cell trafficking, with a focus on imaging-based read-outs. An incisive setback occurred in 2013 when we experienced issues with membrane manufacturing. Cell culture failure resulted from a formulation change in transmigration membranes, the core of our co-culture system, after the supplying company was sold. During our efforts to solve the problem, transmigration experiments were at a standstill.
Projektbezogene Publikationen (Auswahl)
- Development, maintenance and disruption of the blood-brain barrier. Nat. Med. 19(12), 1584-1596 (2013)
Obermeier B., Daneman R., Ransohoff R.M.
- An in vitro blood-brain barrier model combining shear stress and endothelial cell/astrocyte co-culture. J. Neurosci. Methods 232, 165-172(2014)
Takeshita Y., Obermeier B., Cotleur A., Sano Y., Kanda T., Ransohoff R.M.
(Siehe online unter https://doi.org/10.1016/j.jneumeth.2014.05.013)
