Abstract
Increased vascular permeability is the hallmark of inflammation. Here, we describe three methods to assess vascular permeability in cell culture: (1) Impedance measurements of endothelial cell monolayers that allow to monitor changes in cell shape in real time. (2) Diffusion of fluorescently labeled dextran across endothelial cell monolayers to identify openings large enough for bulky molecules. (3) Transmigration of neutrophils through confluent endothelial cell monolayers to study one major process that increases endothelial permeability in inflammation.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Medzhitov R (2008) Origin and physiological roles of inflammation. Nature 454:428–435
Dudek SM, Garcia JG (2001) Cytoskeletal regulation of pulmonary vascular permeability. J Appl Physiol 91:1487–1500
Vandenbroucke E, Mehta D, Minshall R, Malik AB (2008) Regulation of endothelial junctional permeability. Ann N Y Acad Sci 1123:134–145
Hu Z, Liu Q, Wang P. Electric cell substrate impedance sensor (ECIS) as cell-based biosensors. In: Liu Q, Wang P, editors. Cell-based biosensors: Principles and Applications. Boston, London: Artech House Inc., 2009: 151–178.
Shasby DM, Shasby SS (1985) Active transendothelial transport of albumin. Interstitium to lumen. Circ Res 57:903–908
Springer TA (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301–314
Ludwig A, Weber C (2007) Transmembrane chemokines: versatile ‘special agents’ in vascular inflammation. Thromb Haemost 97:694–703
Kuijpers TW, Hakkert BC, Hart MH, Roos D (1992) Neutrophil migration across monolayers of cytokine-prestimulated endothelial cells: a role for platelet-activating factor and IL-8. J Cell Biol 117:565–572
Jaffe EA, Nachman RL, Becker CG, Minick CR (1973) Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52:2745–2756
Abel S, Hundhausen C, Mentlein R, Schulte A, Berkhout TA, Broadway N, Hartmann D, Sedlacek R, Dietrich S, Muetze B, Schuster B, Kallen KJ, Saftig P, Rose-John S, Ludwig A (2004) The transmembrane CXC-chemokine ligand 16 is induced by IFN-gamma and TNF-alpha and shed by the activity of the disintegrin-like metalloproteinase ADAM10. J Immunol 172:6362–6372
Koenen RR, Pruessmeyer J, Soehnlein O, Fraemohs L, Zernecke A, Schwarz N, Reiss K, Sarabi A, Lindbom L, Hackeng TM, Weber C, Ludwig A (2009) Regulated release and functional modulation of junctional adhesion molecule A by disintegrin metalloproteinases. Blood 113:4799–4809
Boyum A (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl 97:77–89.:77–89
Creamer HR, Gabler WL, Bullock WW (1983) Endogenous component chemotactic assay (ECCA). Inflammation 7:321–329
Ludwig A, Petersen F, Zahn S, Gotze O, Schroder JM, Flad HD, Brandt E (1997) The CXC-chemokine neutrophil-activating peptide-2 induces two distinct optima of neutrophil chemotaxis by differential interaction with interleukin-8 receptors CXCR-1 and CXCR-2. Blood 90:4588–4597
Shasby DM, Shasby SS (1986) Effects of calcium on transendothelial albumin transfer and electrical resistance. J Appl Physiol 60:71–79
Lindner K, Uhlig U, Uhlig S (2005) Ceramide alters endothelial cell permeability by a nonapoptotic mechanism. Br J Pharmacol 145:132–140
Lacorre DA, Baekkevold ES, Garrido I, Brandtzaeg P, Haraldsen G, Amalric F, Girard JP (2004) Plasticity of endothelial cells: rapid dedifferentiation of freshly isolated high endothelial venule endothelial cells outside the lymphoid tissue microenvironment. Blood 103:4164–4172
Heydarkhan-Hagvall S, Chien S, Nelander S, Li YC, Yuan S, Lao J, Haga JH, Lian I, Nguyen P, Risberg B, Li YS (2006) DNA microarray study on gene expression profiles in co-cultured endothelial and smooth muscle cells in response to 4- and 24-h shear stress. Mol Cell Biochem 281:1–15
Uhlig S, Lindner K. Novel mechanisms of endothelial cell permeability in the lung. Lessons from studies with intact lungs and animals. In: Tooke J, Shore A, Whatmore J, editors. The microcirculation and vascular biology. Bologna: Monduzi Editore, 2002: 221–234.
Fujiwara K (2003) Mechanical stresses keep endothelial cells healthy: beneficial effects of a physiological level of cyclic stretch on endothelial barrier function. Am J Physiol Lung Cell Mol Physiol 285:L782–L784
Gratton JP, Bernatchez P, Sessa WC (2004) Caveolae and caveolins in the cardiovascular system. Circ Res 94:1408–1417
Hall CN, Garthwaite J (2009) What is the real physiological NO concentration in vivo? Nitric Oxide 21:92–103
Kuebler WM, Yang Y, Samapati R, Uhlig S (2010) Vascular barrier regulation by PAF, ceramide, caveolae, and NO – an intricate signaling network with discrepant effects in the pulmonary and systemic vasculature. Cell Physiol Biochem 26:29–40
Molema G, Mesander G, Kroesen BJ, Helfrich W, Meijer DK, de Leij LF (1998) Analysis of in vitro lymphocyte adhesion and transendothelial migration by fluorescent-beads-based flow cytometric cell counting. Cytometry 32:37–43
Acknowledgments
This work was supported by the IZKF Aachen and by the DFG, SFB 542, Projects A12 and C16.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Ludwig, A., Sommer, A., Uhlig, S. (2011). Assessment of Endothelial Permeability and Leukocyte Transmigration in Human Endothelial Cell Monolayers. In: Turksen, K. (eds) Permeability Barrier. Methods in Molecular Biology, vol 763. Humana Press. https://doi.org/10.1007/978-1-61779-191-8_22
Download citation
DOI: https://doi.org/10.1007/978-1-61779-191-8_22
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-190-1
Online ISBN: 978-1-61779-191-8
eBook Packages: Springer Protocols