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Detection of Bidirectional Signaling During Integrin Activation and Neutrophil Adhesion

  • Stuart M. Altman
  • Neha Dixit
  • Scott I. Simon
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1124)

Abstract

Neutrophil arrest and migration on inflamed endothelium is dependent upon a conformational shift in CD11a/CD18 (LFA-1) from a low to high affinity and clustered state which determines the strength and lifetime of bond formation with intracellular adhesion molecule 1 (ICAM-1). Cytoskeletal adaptor proteins kindlin-3 and talin-1 anchor clustered LFA-1 to the cytoskeleton and support the transition from neutrophil rolling to arrest. We employ microfluidic flow channels and total internal reflection fluorescence microscopy to evaluate the spatiotemporal regulation of LFA-1 affinity and bond formation that facilitate the transition from neutrophil rolling to arrest. Methodology is presented to correlate the relationship between integrin conformation, bond formation with ICAM-1, and cytoskeletal engagement and adhesion strengthening necessary to achieve a migratory phenotype.

Keywords

Integrin Selectin ICAM-1 Intercellular adhesion molecule-1 Leukocyte function associated protein-1 ( LFA-1) Macrophage protein-1 (Mac-1) Adhesion strengthening G-protein coupled receptors Cytoskeleton 

Notes

Acknowledgements

This work was supported by National Institutes of Health (NIH) grant AI472294 to S.I.S.

References

  1. 1.
    Campbell JJ, Hedrick J, Zlotnik A et al (1998) Chemokines and the arrest of lymphocytes rolling under flow conditions. Science 279: 381–384PubMedCrossRefGoogle Scholar
  2. 2.
    Ley K (2002) Integration of inflammatory signals by rolling neutrophils. Immunol Rev 186: 8–18PubMedCrossRefGoogle Scholar
  3. 3.
    Simon SI, Green CE (2005) Molecular mechanics and dynamics of leukocyte recruitment during inflammation. Annu Rev Biomed Eng 7:151–185PubMedCrossRefGoogle Scholar
  4. 4.
    Dwir O, Kansas GS, Alon R (2001) Cytoplasmic anchorage of L-selectin controls leukocyte capture and rolling by increasing the mechanical stability of the selectin tether. J Cell Biol 155: 145–156PubMedCrossRefGoogle Scholar
  5. 5.
    Taylor AD, Neelamegham S, Hellums JD et al (1996) Molecular dynamics of the transition from L-selectin- to beta 2-integrin-dependent neutrophil adhesion under defined hydrodynamic shear. Biophys J 71:3488–3500PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Thomas WE, Trintchina E, Forero M et al (2002) Bacterial adhesion to target cells enhanced by shear force. Cell 109:913–923PubMedCrossRefGoogle Scholar
  7. 7.
    Zhu C, McEver RP (2005) Catch bonds: physical models and biological functions. Mol Cell Biomech 2:91–104PubMedGoogle Scholar
  8. 8.
    McDonough DB, McIntosh FA, Spanos C et al (2004) Cooperativity between selectins and beta2-integrins define neutrophil capture and stable adhesion in shear flow. Ann Biomed Eng 32:1179–1192PubMedCrossRefGoogle Scholar
  9. 9.
    Zarbock A, Lowell CA, Ley K (2007) Spleen tyrosine kinase Syk is necessary for E-selectin-induced alpha(L)beta(2) integrin-mediated rolling on intercellular adhesion molecule-1. Immunity 26:773–783PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Simon SI, Hu Y, Vestweber D et al (2000) Neutrophil tethering on E-selectin activates beta 2 integrin binding to ICAM-1 through a mitogen-activated protein kinase signal transduction pathway. J Immunol 164:4348–4358PubMedGoogle Scholar
  11. 11.
    Green CE, Schaff UY, Sarantos MR et al (2006) Dynamic shifts in LFA-1 affinity regulate neutrophil rolling, arrest, and transmigration on inflamed endothelium. Blood 107: 2101–2111PubMedCrossRefGoogle Scholar
  12. 12.
    Alon R, Feigelson S (2002) From rolling to arrest on blood vessels: leukocyte tap dancing on endothelial integrin ligands and chemokines at sub-second contacts. Semin Immunol 14: 93–104PubMedCrossRefGoogle Scholar
  13. 13.
    Schaff UY, Yamayoshi I, Tse T et al (2008) Calcium flux in neutrophils synchronizes beta2 integrin adhesive and signaling events that guide inflammatory recruitment. Ann Biomed Eng 36:632–646PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Beals CR, Edwards AC, Gottschalk RJ et al (2001) CD18 activation epitopes induced by leukocyte activation. J Immunol 167: 6113–6122PubMedGoogle Scholar
  15. 15.
    Constantin G, Majeed M, Giagulli C et al (2000) Chemokines trigger immediate beta2 integrin affinity and mobility changes: differential regulation and roles in lymphocyte arrest under flow. Immunity 13:759–769PubMedCrossRefGoogle Scholar
  16. 16.
    Kim M, Carman CV, Yang W et al (2004) The primacy of affinity over clustering in regulation of adhesiveness of the integrin αLβ2. J Cell Biol 167:1241–1253PubMedCrossRefGoogle Scholar
  17. 17.
    Sarantos MR, Raychaudhuri S, Lum AF et al (2005) Leukocyte function-associated antigen 1-mediated adhesion stability is dynamically regulated through affinity and valency during bond formation with intercellular adhesion molecule-1. J Biol Chem 280:28290–28298PubMedCrossRefGoogle Scholar
  18. 18.
    Bachmann MF, Kopf M, Marsland BJ (2006) Chemokines: more than just road signs. Nat Rev Immunol 6:159–164PubMedCrossRefGoogle Scholar
  19. 19.
    Zarbock A, Deem TL, Burcin TL et al (2007) Galphai2 is required for chemokine-induced neutrophil arrest. Blood 110:3773–3779PubMedCrossRefGoogle Scholar
  20. 20.
    Alon R, Ley K (2008) Cells on the run: shear-regulated integrin activation in leukocyte rolling and arrest on endothelial cells. Curr Opin Cell Biol 20:525–532PubMedCrossRefGoogle Scholar
  21. 21.
    Lum AF, Green CE, Lee GR et al (2002) Dynamic regulation of LFA-1 activation and neutrophil arrest on intercellular adhesion molecule 1 (ICAM-1) in shear flow. J Biol Chem 277:20660–20670PubMedCrossRefGoogle Scholar
  22. 22.
    Dixit N, Simon SI (2012) Chemokines, selectins and intracellular calcium flux: temporal and spatial cues for leukocyte arrest. Front Immunol 3:188PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Dixit N, Yamayoshi I, Nazarian A et al (2011) Migrational guidance of neutrophils is mechanotransduced via high-affinity LFA-1 and calcium flux. J Immunol 187:472–481PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Alon R, Dustin ML (2007) Force as a facilitator of integrin conformational changes during leukocyte arrest on blood vessels and antigen-presenting cells. Immunity 26:17–27PubMedCrossRefGoogle Scholar
  25. 25.
    Schreiber TH, Shinder V, Cain DW et al (2007) Shear flow-dependent integration of apical and subendothelial chemokines in T-cell transmigration: implications for locomotion and the multistep paradigm. Blood 109: 1381–1386PubMedCrossRefGoogle Scholar
  26. 26.
    Mitchon LN, White JM (2006) Growth and analysis of octadecylsiloxane monolayers on Al2O3 (0001). Langmuir 22:6549–6554PubMedCrossRefGoogle Scholar
  27. 27.
    Schaff UY, Dixit N, Procyk E et al (2009) Orai1 regulates intracellular calcium, arrest, and shape polarization during neutrophil recruitment in shear flow. Blood 115:657–666PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Stuart M. Altman
    • 1
  • Neha Dixit
    • 1
  • Scott I. Simon
    • 1
  1. 1.Department of Biomedical EngineeringUniversity of CaliforniaDavisUSA

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