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High-Resolution Fluorescence Microscopy to Study Transendothelial Migration

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Integrin and Cell Adhesion Molecules

Part of the book series: Methods in Molecular Biology ((MIMB,volume 757))

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Abstract

Immune system functions rely heavily on the ability of immune cells (i.e., blood leukocyte) to traffic throughout the body as they conduct immune surveillance and respond to pathogens. A monolayer of vascular endothelial cells (i.e., the “endothelium”) provides a critical, selectively permeable barrier between two principal compartments of the body: the blood circulation and the tissue. Thus, knowledge of the basic mechanisms by which leukocytes migrate across the endothelium (i.e., undergo “transendothelial migration”; TEM) is critical for understanding immune system function. Cultured endothelial cell monolayers, used in combination with isolated blood leukocytes, provide a basis for highly useful in vitro models for study of TEM. When used in conjunction with high spatial and temporal resolution imaging approaches, such models have begun to reveal complex and dynamic cell behaviors in leukocytes and endothelial cells that ultimately determine TEM efficiency. In this chapter, we provide protocols for setting up a basic in vitro TEM system and for conducting high-resolution dynamic live-cell and three-dimensional fixed-cell imaging of TEM.

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References

  1. von Andrian, U. H., and Mackay, C. R. (2000) T-cell function and migration. Two sides of the same coin. N. Engl. J. Med. 343, 1020–1034.

    Article  Google Scholar 

  2. Bazzoni, G., and Dejana, E. (2004) Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiol. Rev. 84, 869–901.

    Article  PubMed  CAS  Google Scholar 

  3. Pepper, M.S., and Skobe, M. (2003) Lymphatic endothelium: morphological, molecular and functional properties. J. Cell Biol. 163, 209–213.

    Article  PubMed  CAS  Google Scholar 

  4. Baluk, P., Fuxe, J., Hashizume, H., Romano, T., Lashnits, E., Butz, S., Vestweber, D., Corada, M., Molendini, C., Dejana, E., and McDonald, D. M. (2007) Functionally specialized junctions between endothelial cells of lymphatic vessels. J. Exp. Med. 204, 2349–2362.

    Article  PubMed  CAS  Google Scholar 

  5. Ley, K., Laudanna, C., Cybulsky, M. I., and Nourshargh, S. (2007) Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Immunol. 7, 678–689.

    Article  PubMed  CAS  Google Scholar 

  6. Huttenlocher, A., and Poznansky, M. C. (2008) Reverse leukocyte migration can be attractive or repulsive. Trend. Cell Biol. 18, 298–306.

    Article  CAS  Google Scholar 

  7. Springer, T. A. (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76, 301–314.

    Article  PubMed  CAS  Google Scholar 

  8. Luscinskas, F. W., Kansas, G. S., Ding, H., Pizcueta, P., Schleiffenbaum, B. E., Tedder, T. F., and Gimbrone, M. A., Jr. (1994) Monocyte rolling, arrest and spreading on IL-4-activated vascular endothelium under flow is mediated via sequential action of L-selectin, beta 1-integrins, and beta 2-integrins. J. Cell Biol. 125, 1417–1427.

    Article  PubMed  CAS  Google Scholar 

  9. Butcher, E. C. (1991) Leukocyte-endothelial cell recognition: Three (or more) steps to specificity and diversity. Cell 67, 1033–1036.

    Article  PubMed  CAS  Google Scholar 

  10. Carman, C. V., and Springer, T. A. (2003) Integrin avidity regulation: are changes in affinity and conformation underemphasized? Curr. Opin. Cell Biol. 15, 547–556.

    Article  PubMed  CAS  Google Scholar 

  11. Luo, B. H., Carman, C. V., and Springer, T. A. (2007) Structural basis of integrin regulation and signaling. Annu. Rev. Immunol. 25, 619–647.

    Article  PubMed  CAS  Google Scholar 

  12. Schenkel, A. R., Mamdouh, Z., and Muller, W. A. (2004) Locomotion of monocytes on endothelium is a critical step during extravasation. Nat. Immunol. 5, 393–400.

    Article  PubMed  CAS  Google Scholar 

  13. Phillipson, M., Heit, B., Colarusso, P., Liu, L., Ballantyne, C. M., and Kubes, P. (2006) Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade. J. Exp. Med. 203, 2569–2575.

    Article  PubMed  CAS  Google Scholar 

  14. Luscinskas, F. W., Ma, S., Nusrat, A., Parkos, C. A., and Shaw, S. K. (2002) Leukocyte transendothelial migration: a junctional affair. Semin. Immunol. 14, 105–113.

    Article  PubMed  CAS  Google Scholar 

  15. Muller, W. A. (2003) Leukocyte-endothelial-cell interactions in leukocyte transmigration and the inflammatory response. Trend. Immunol. 24, 327–334.

    CAS  Google Scholar 

  16. Muller, W. A. (2001) Migration of leukocytes across endothelial junctions: some concepts and controversies. Microcirculation 8, 181–193.

    PubMed  CAS  Google Scholar 

  17. Burns, A. R., Smith, C. W., and Walker, D. C. (2003) Unique structural features that influence neutrophil emigration into the lung. Physiol. Rev. 83, 309–336.

    PubMed  CAS  Google Scholar 

  18. Williamson, J. R., and Grisham, J. W. (1960) Leucocytic emigration from inflamed capillaries. Nature 188, 1203.

    Article  PubMed  CAS  Google Scholar 

  19. Williamson, J. R., and Grisham, J. W. (1961) Electron microscopy of leukocytic margination and emigration in acute inflammation in dog pancreas. Am. J. Pathol. 39, 239–256.

    PubMed  CAS  Google Scholar 

  20. Marchesi, V. T., and Gowans, J. L. (1964) The Migration of Lymphocytes through the Endothelium of Venules in Lymph Nodes: an Electron Microscope Study. Proc. R. Soc. Lond. B. Biol. Sci. 159, 283–290.

    Article  PubMed  CAS  Google Scholar 

  21. Sage, P. T., and Carman, C. V. (2009) Settings and Mechanisms for Trans-cellular Diapedesis. Front. Biosci. 4, 5066–5083.

    Article  Google Scholar 

  22. Carman, C. V., and Springer, T. A. (2008) Trans-cellular migration: cell-cell contacts get intimate. Curr. Opin. Cell Biol. 20, 533–540.

    Article  PubMed  CAS  Google Scholar 

  23. Linder, S., and Aepfelbacher, M. (2003) Podosomes: adhesion hot-spots of invasive cells. Trend. Cell Biol. 13, 376–385.

    Article  CAS  Google Scholar 

  24. Buccione, R., Orth, J. D., and McNiven, M. A. (2004) Foot and mouth: podosomes, invadopodia and circular dorsal ruffles. Nat. Rev. Mol. Cell Biol. 5, 647–657.

    Article  PubMed  CAS  Google Scholar 

  25. Yamaguchi, H., Wyckoff, J., and Condeelis, J. (2005) Cell migration in tumors. Curr. Opin. Cell Biol. 17, 559–564.

    Article  PubMed  CAS  Google Scholar 

  26. Carman, C. V., Sage, P. T., Sciuto, T. E., de la Fuente, M. A., Geha, R. S., Ochs, H. D., Dvorak, H. F., Dvorak, A. M., and Springer, T. A. (2007) Transcellular diapedesis is initiated by invasive podosomes. Immunity 26, 784–797.

    Article  PubMed  CAS  Google Scholar 

  27. Hidalgo, A., and Frenette, P. S. (2007) Leukocyte podosomes sense their way through the endothelium. Immunity 26, 753–755.

    Article  PubMed  CAS  Google Scholar 

  28. Carman, C. V. (2009) Mechanisms for transcellular diapedesis: probing and pathfinding by “invadosome-like protrusions”. J. Cell Sci. 122, 3025–3035.

    Article  PubMed  CAS  Google Scholar 

  29. Gerard, A., van der Kammen, R. A., Janssen, H., Ellenbroek, S. I., and Collard, J. G. (2009) The Rac activator Tiam1 controls efficient T-cell trafficking and route of transendothelial migration. Blood 113, 6138–6147.

    Article  PubMed  CAS  Google Scholar 

  30. Wojciak-Stothard, B., Williams, L., and Ridley, A. J. (1999) Monocyte adhesion and spreading on human endothelial cells is dependent on Rho-regulated receptor clustering. J. Cell Biol. 145, 1293–1307.

    Article  PubMed  CAS  Google Scholar 

  31. Barreiro, O., Yanez-Mo, M., Serrador, J. M., Montoya, M. C., Vicente-Manzanares, M., Tejedor, R., Furthmayr, H., and Sanchez-Madrid, F. (2002) Dynamic interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes. J. Cell Biol. 157, 1233–1245.

    Article  PubMed  CAS  Google Scholar 

  32. Carman, C. V., Jun, C. D., Salas, A., and Springer, T. A. (2003) Endothelial cells proactively form microvilli-like membrane ­projections upon intercellular adhesion molecule 1 engagement of leukocyte LFA-1. J. Immunol. 171, 6135–6144.

    PubMed  CAS  Google Scholar 

  33. Carman, C. V., and Springer, T. A. (2004) A transmigratory cup in leukocyte diapedesis both through individual vascular endothelial cells and between them. J. Cell Biol. 167, 377–388.

    Article  PubMed  CAS  Google Scholar 

  34. Barreiro, O., Yanez-Mo, M., Sala-Valdes, M., Gutierrez-Lopez, M. D., Ovalle, S., Higginbottom, A., Monk, P. N., Cabanas, C., and Sanchez-Madrid, F. (2005) Endothelial tetraspanin microdomains regulate leukocyte firm adhesion during extravasation. Blood 105, 2852–2861.

    Article  PubMed  CAS  Google Scholar 

  35. Millan, J., Hewlett, L., Glyn, M., Toomre, D., Clark, P., and Ridley, A. J. (2006) Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola- and F-actin-rich domains. Nat. Cell Biol. 8, 113–123.

    Article  PubMed  CAS  Google Scholar 

  36. Nieminen, M., Henttinen, T., Merinen, M., Marttila-Ichihara, F., Eriksson, J. E., and Jalkanen, S. (2006) Vimentin function in lymphocyte adhesion and transcellular migration. Nat. Cell Biol. 8, 156–162.

    Article  PubMed  CAS  Google Scholar 

  37. van Buul, J. D., Allingham, M. J., Samson, T., Meller, J., Boulter, E., Garcia-Mata, R., and Burridge, K. (2007) RhoG regulates endothelial apical cup assembly downstream from ICAM1 engagement and is involved in leukocyte trans-endothelial migration. J. Cell Biol. 178, 1279–1293.

    Article  PubMed  Google Scholar 

  38. Riethmuller, C., Nasdala, I., and Vestweber, D. (2008) Nano-surgery at the leukocyte-endothelial docking site. Pflugers Arch. 456, 71–81.

    Article  PubMed  Google Scholar 

  39. Barreiro, O., Zamai, M., Yanez-Mo, M., Tejera, E., Lopez-Romero, P., Monk, P. N., Gratton, E., Caiolfa, V. R., and Sanchez-Madrid, F. (2008) Endothelial adhesion receptors are recruited to adherent leukocytes by inclusion in preformed tetraspanin nanoplatforms. J. Cell Biol. 183, 527–542.

    Article  PubMed  CAS  Google Scholar 

  40. Kanters, E., van Rijssel, J., Hensbergen, P. J., Hondius, D., Mul, F. P., Deelder, A. M., Sonnenberg, A., van Buul, J. D., and Hordijk, P. L. (2008) Filamin B mediates ICAM-1-driven leukocyte transendothelial migration. J. Biol. Chem. 283, 31830–31839.

    Article  PubMed  CAS  Google Scholar 

  41. Cayrol, R., Wosik, K., Berard, J. L., Dodelet-Devillers, A., Ifergan, I., Kebir, H., Haqqani, A. S., Kreymborg, K., Krug, S., Moumdjian, R., Bouthillier, A., Becher, B., Arbour, N., David, S., Stanimirovic, D., and Prat, A. (2008) Activated leukocyte cell adhesion molecule promotes leukocyte trafficking into the central nervous system. Nat. Immunol. 9, 137–145.

    Article  PubMed  CAS  Google Scholar 

  42. Dittmar, S., Harms, H., Runkler, N., Maisner, A., Kim, K. S., and Schneider-Schaulies, J. (2008) Measles virus-induced block of transendothelial migration of T lymphocytes and infection-mediated virus spread across endothelial cell barriers. J. Virol. 82, 11273–11282.

    Article  PubMed  CAS  Google Scholar 

  43. Rohlena, J., Volger, O. L., van Buul, J. D., Hekking, L. H., van Gils, J. M., Bonta, P. I., Fontijn, R. D., Post, J. A., Hordijk, P. L., and Horrevoets, A. J. (2009) Endothelial CD81 is a marker of early human atherosclerotic plaques and facilitates monocyte adhesion. Cardiovasc. Res. 81, 187–196.

    Article  PubMed  CAS  Google Scholar 

  44. Beltman, J. B., Maree, A. F., and de Boer, R. J. (2009) Analysing immune cell migration. Nat. Rev. Immunol. 9, 789–798.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by the Arthritis Foundation, American Heart Association, and Roche Organ Transplant Research Foundation.

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Authors and Affiliations

  1. Center for Vascular Biology Research, Division of Molecular and Vascular Medicine, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Christopher V. Carman

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  1. Christopher V. Carman
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Correspondence to Christopher V. Carman .

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Editors and Affiliations

  1. Immune Disease Institute, Harvard Medical School, Longwood Avenue 200, Boston, 02115, Massachusetts, USA

    Motomu Shimaoka

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Carman, C.V. (2011). High-Resolution Fluorescence Microscopy to Study Transendothelial Migration. In: Shimaoka, M. (eds) Integrin and Cell Adhesion Molecules. Methods in Molecular Biology, vol 757. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-166-6_15

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  • DOI: https://doi.org/10.1007/978-1-61779-166-6_15

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-165-9

  • Online ISBN: 978-1-61779-166-6

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