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Imaging Interactions Between the Immune and Cardiovascular Systems In Vivo by Multiphoton Microscopy

  • Owain R. Millington
  • James M. Brewer
  • Paul Garside
  • Pasquale Maffia
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 616)

Abstract

Several recent studies in immunology have used multiphoton laser-scanning microscopy to visualise the induction of an immune response in real time in vivo. These experiments are illuminating the cellular and molecular interactions involved in the induction, maintenance and regulation of immune responses. Similar approaches are being applied in cardiovascular research where there is an increasing body of evidence to support a significant role for the adaptive immune system in vascular disease. As such, we have begun to dissect the role of T lymphocytes in atherosclerosis in real time in vivo. Here, we provide step-by-step guides to the various stages involved in visualising the migration of T cells within a lymph node and their infiltration into inflamed tissues such as atherosclerotic arteries. These methods provide an insight into the mechanisms involved in the activation and function of immune cells in vivo.

Key words

Brachiocephalic artery imaging lymph node multiphoton microscopy T cells 

References

  1. 1.
    Robertson AK, Hansson GK. (2006) T cells in atherogenesis: for better or for worse? Arterioscler Thromb Vasc Biol 26, 2421–32.PubMedCrossRefGoogle Scholar
  2. 2.
    Hansson GK, Libby P. (2006) The immune response in atherosclerosis: a double-edged sword. Nat Rev Immunol 6, 508–19.PubMedCrossRefGoogle Scholar
  3. 3.
    Hugues S, Fetler L, Bonifaz L, Helft J, Amblard F, Amigorena S. (2004) Distinct T cell dynamics in lymph nodes during the induction of tolerance and immunity. Nat Immunol 5, 1235–42.PubMedCrossRefGoogle Scholar
  4. 4.
    Shakhar G, Lindquist RL, Skokos D, Dudziak D, Huang JH, Nussenzweig MC, Dustin ML. (2005) Stable T cell-dendritic cell interactions precede the development of both tolerance and immunity in vivo. Nat Immunol 6, 707–14.PubMedCrossRefGoogle Scholar
  5. 5.
    Zinselmeyer BH, Dempster J, Gurney AM, Wokosin D, Miller M, Ho H, Millington OR, Smith KM, Rush CM, Parker I, Cahalan M, Brewer JM, Garside P. (2005) In situ characterization of CD4+ T cell behaviour in mucosal and systemic lymphoid tissues during the induction of oral priming and tolerance. J Exp Med 201, 1815–23.PubMedCrossRefGoogle Scholar
  6. 6.
    Millington OR, Gibson VB, Rush CM, Zinselmeyer BH, Phillips RS, Garside P, Brewer JM. (2007) Malaria impairs T cell clustering and immune priming despite normal signal 1 from dendritic cells. PLoS Pathog 3, 1380–87.PubMedCrossRefGoogle Scholar
  7. 7.
    Miller MJ, Wei SH, Parker I, Cahalan MD. (2002) Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296, 1869–73.PubMedCrossRefGoogle Scholar
  8. 8.
    Mempel TR, Henrickson SE, Von Andrian UH. (2004) T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature 427, 154–59.PubMedCrossRefGoogle Scholar
  9. 9.
    Miller MJ, Hejazi AS, Wei SH, Cahalan MD, Parker I. (2004) T cell repertoire scanning is promoted by dynamic dendritic cell behavior and random T cell motility in the lymph node. Proc Natl Acad Sci USA 101, 998–1003.PubMedCrossRefGoogle Scholar
  10. 10.
    Celli S, Garcia Z, Bousso P. (2005) CD4 T cells integrate signals delivered during successive DC encounters in vivo. J Exp Med 202, 1271–78.PubMedCrossRefGoogle Scholar
  11. 11.
    Schneider H, Downey J, Smith A, Zinselmeyer BH, Rush C, Brewer JM, Wei B, Hogg N, Garside P, Rudd CE. (2006) Reversal of the TCR stop signal by CTLA-4. Science 313, 1972–75.PubMedCrossRefGoogle Scholar
  12. 12.
    Millington OR, Zinselmeyer BH, Brewer JM, Garside P, Rush CM. (2007) Lymphocyte tracking and interactions in secondary lymphoid organs. Inflamm Res 56, 391–401.PubMedCrossRefGoogle Scholar
  13. 13.
    Okada T, Miller MJ, Parker I, Krummel MF, Neighbors M, Hartley SB, O’Garra A, Cahalan MD, Cyster JG. (2005) Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. PLoS Biol 3, e150.PubMedCrossRefGoogle Scholar
  14. 14.
    Garside P, Brewer JM. (2008) Real-time imaging of the cellular interactions underlying tolerance, priming, and responses to infection. Immunol Rev 221, 130–46.PubMedCrossRefGoogle Scholar
  15. 15.
    Matheu MP, Parker I, Cahalan MD (2007) Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice. J Vis Exp http://www.jove.com/index/Details.stp?ID=265
  16. 16.
    Maffia P, Zinselmeyer BH, Ialenti A, Kennedy S, Baker AH, McInnes IB, Brewer JM, Garside P. (2007) Images in cardiovascular medicine. Multiphoton microscopy for 3-dimensional imaging of lymphocyte recruitment into apolipoprotein-E-deficient mouse carotid artery. Circulation 115, e326–8.PubMedCrossRefGoogle Scholar
  17. 17.
    Celli S, Lemaitre F, Bousso P. (2007) Real-time manipulation of T cell-dendritic cell interactions in vivo reveals the importance of prolonged contacts for CD4+ T cell activations. Immunity 27, 625–34.PubMedCrossRefGoogle Scholar
  18. 18.
    Galkina E, Kadl A, Sanders J, Varughese D, Sarembock IJ, Ley K. (2006) Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent. J Exp Med 203, 1273–82.PubMedCrossRefGoogle Scholar
  19. 19.
    Matheu MP, Cahalan MD (2007) Isolation of CD4+ T cells from Mouse Lymph Nodes using Miltenyi MACS Purification. J Vis Exp http://www.jove.com/index/Details.stp?ID=409
  20. 20.
    Huang JH, Cárdenas-Navia LI, Caldwell CC, Plumb TJ, Radu CG, Rocha PN, Wilder T, Bromberg JS, Cronstein BN, Sitkovsky M, Dewhirst MW, Dustin ML. (2007) Requirements for T lymphocyte migration in explanted lymph nodes. J Immunol 178, 7747–55.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Owain R. Millington
    • 1
  • James M. Brewer
    • 1
  • Paul Garside
    • 1
  • Pasquale Maffia
    • 2
    • 3
  1. 1.Centre for Biophotonics, Strathclyde Institute of Pharmacy & Biomedical SciencesUniversity of StrathclydeGlasgowUK
  2. 2.Strathclyde Institute of Pharmacy & Biomedical SciencesUniversity of StrathclydeGlasgowUK
  3. 3.Department of Experimental Pharmacology School of Biotechnological SciencesUniversity of Naples Federico IINaplesItaly

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