High-Resolution Fluorescence Microscopy to Study Transendothelial Migration

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

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.

Key words

Fluorescence Microscopy Leukocyte Endothelium Diapedesis Transmigration GFP Imaging Antibody Lymphocyte 

Notes

Acknowledgments

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

References

  1. 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.CrossRefGoogle Scholar
  2. 2.
    Bazzoni, G., and Dejana, E. (2004) Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiol. Rev. 84, 869–901.PubMedCrossRefGoogle Scholar
  3. 3.
    Pepper, M.S., and Skobe, M. (2003) Lymphatic endothelium: morphological, molecular and functional properties. J. Cell Biol. 163, 209–213.PubMedCrossRefGoogle Scholar
  4. 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.PubMedCrossRefGoogle Scholar
  5. 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.PubMedCrossRefGoogle Scholar
  6. 6.
    Huttenlocher, A., and Poznansky, M. C. (2008) Reverse leukocyte migration can be attractive or repulsive. Trend. Cell Biol. 18, 298–306.CrossRefGoogle Scholar
  7. 7.
    Springer, T. A. (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76, 301–314.PubMedCrossRefGoogle Scholar
  8. 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.PubMedCrossRefGoogle Scholar
  9. 9.
    Butcher, E. C. (1991) Leukocyte-endothelial cell recognition: Three (or more) steps to specificity and diversity. Cell 67, 1033–1036.PubMedCrossRefGoogle Scholar
  10. 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.PubMedCrossRefGoogle Scholar
  11. 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.PubMedCrossRefGoogle Scholar
  12. 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.PubMedCrossRefGoogle Scholar
  13. 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.PubMedCrossRefGoogle Scholar
  14. 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.PubMedCrossRefGoogle Scholar
  15. 15.
    Muller, W. A. (2003) Leukocyte-endothelial-cell interactions in leukocyte transmigration and the inflammatory response. Trend. Immunol. 24, 327–334.Google Scholar
  16. 16.
    Muller, W. A. (2001) Migration of leukocytes across endothelial junctions: some concepts and controversies. Microcirculation 8, 181–193.PubMedGoogle Scholar
  17. 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.PubMedGoogle Scholar
  18. 18.
    Williamson, J. R., and Grisham, J. W. (1960) Leucocytic emigration from inflamed capillaries. Nature 188, 1203.PubMedCrossRefGoogle Scholar
  19. 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.PubMedGoogle Scholar
  20. 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.PubMedCrossRefGoogle Scholar
  21. 21.
    Sage, P. T., and Carman, C. V. (2009) Settings and Mechanisms for Trans-cellular Diapedesis. Front. Biosci. 4, 50665083.CrossRefGoogle Scholar
  22. 22.
    Carman, C. V., and Springer, T. A. (2008) Trans-cellular migration: cell-cell contacts get intimate. Curr. Opin. Cell Biol. 20, 533–540.PubMedCrossRefGoogle Scholar
  23. 23.
    Linder, S., and Aepfelbacher, M. (2003) Podosomes: adhesion hot-spots of invasive cells. Trend. Cell Biol. 13, 376–385.CrossRefGoogle Scholar
  24. 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.PubMedCrossRefGoogle Scholar
  25. 25.
    Yamaguchi, H., Wyckoff, J., and Condeelis, J. (2005) Cell migration in tumors. Curr. Opin. Cell Biol. 17, 559–564.PubMedCrossRefGoogle Scholar
  26. 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.PubMedCrossRefGoogle Scholar
  27. 27.
    Hidalgo, A., and Frenette, P. S. (2007) Leukocyte podosomes sense their way through the endothelium. Immunity 26, 753–755.PubMedCrossRefGoogle Scholar
  28. 28.
    Carman, C. V. (2009) Mechanisms for transcellular diapedesis: probing and pathfinding by “invadosome-like protrusions”. J. Cell Sci. 122, 3025–3035.PubMedCrossRefGoogle Scholar
  29. 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.PubMedCrossRefGoogle Scholar
  30. 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.PubMedCrossRefGoogle Scholar
  31. 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.PubMedCrossRefGoogle Scholar
  32. 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.PubMedGoogle Scholar
  33. 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.PubMedCrossRefGoogle Scholar
  34. 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.PubMedCrossRefGoogle Scholar
  35. 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.PubMedCrossRefGoogle Scholar
  36. 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.PubMedCrossRefGoogle Scholar
  37. 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.PubMedCrossRefGoogle Scholar
  38. 38.
    Riethmuller, C., Nasdala, I., and Vestweber, D. (2008) Nano-surgery at the leukocyte-endothelial docking site. Pflugers Arch. 456, 71–81.PubMedCrossRefGoogle Scholar
  39. 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.PubMedCrossRefGoogle Scholar
  40. 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.PubMedCrossRefGoogle Scholar
  41. 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.PubMedCrossRefGoogle Scholar
  42. 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.PubMedCrossRefGoogle Scholar
  43. 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.PubMedCrossRefGoogle Scholar
  44. 44.
    Beltman, J. B., Maree, A. F., and de Boer, R. J. (2009) Analysing immune cell migration. Nat. Rev. Immunol. 9, 789–798.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Center for Vascular Biology Research, Division of Molecular and Vascular Medicine, Department of MedicineBeth Israel Deaconess Medical Center, Harvard Medical SchoolBostonUSA

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