Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

‘Slings’ enable neutrophil rolling at high shear

Subjects

Abstract

Most leukocytes can roll along the walls of venules at low shear stress (1 dyn cm−2), but neutrophils have the ability to roll at tenfold higher shear stress in microvessels in vivo1,2. The mechanisms involved in this shear-resistant rolling are known to involve cell flattening3 and pulling of long membrane tethers at the rear4,5,6. Here we show that these long tethers do not retract as postulated6,7, but instead persist and appear as ‘slings’ at the front of rolling cells. We demonstrate slings in a model of acute inflammation in vivo and on P-selectin in vitro, where P-selectin-glycoprotein-ligand-1 (PSGL-1) is found in discrete sticky patches whereas LFA-1 is expressed over the entire length on slings. As neutrophils roll forward, slings wrap around the rolling cells and undergo a step-wise peeling from the P-selectin substrate enabled by the failure of PSGL-1 patches under hydrodynamic forces. The ‘step-wise peeling of slings’ is distinct from the ‘pulling of tethers’ reported previously4,5,6,8. Each sling effectively lays out a cell-autonomous adhesive substrate in front of neutrophils rolling at high shear stress during inflammation.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Rolling neutrophils form slings.
Figure 2: Sling formation, length and force.
Figure 3: Neutrophil rolling is stabilized by step-wise peeling of slings.
Figure 4: Slings enable LFA-1-ICAM-2 interactions in trans.

References

  1. Firrell, J. C. & Lipowsky, H. H. Leukocyte margination and deformation in mesenteric venules of rat. Am. J. Physiol. Heart Circ. Physiol. 256, H1667–H1674 (1989)

    CAS  Article  Google Scholar 

  2. Sundd, P., Pospieszalska, M. K., Cheung, L. S., Konstantopoulos, K. & Ley, K. Biomechanics of leukocyte rolling. Biorheology 48, 1–35 (2011)

    PubMed  PubMed Central  Google Scholar 

  3. Damiano, E. R., Westheider, J., Tozeren, A. & Ley, K. Variation in the velocity, deformation, and adhesion energy density of leukocytes rolling within venules. Circ. Res. 79, 1122–1130 (1996)

    CAS  Article  PubMed  Google Scholar 

  4. Sundd, P. et al. Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling. Nature Methods 7, 821–824 (2010)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Ramachandran, V., Williams, M., Yago, T., Schmidtke, D. W. & McEver, R. P. Dynamic alterations of membrane tethers stabilize leukocyte rolling on P-selectin. Proc. Natl Acad. Sci. USA 101, 13519–13524 (2004)

    CAS  ADS  Article  PubMed  PubMed Central  Google Scholar 

  6. Schmidtke, D. W. & Diamond, S. L. Direct observation of membrane tethers formed during neutrophil attachment to platelets or P-selectin under physiological flow. J. Cell Biol. 149, 719–730 (2000)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Shao, J.-Y. in Leukocyte Adhesion (ed. Ley, K. ) Ch. 2, 25–45 (Academic, 2009).

  8. Pospieszalska, M. K., Lasiecka, I. & Ley, K. Cell protrusions and tethers: a unified approach. Biophys. J. 100, 1697–1707 (2011)

    ADS  Article  PubMed  PubMed Central  Google Scholar 

  9. Hocdé, S. A., Hyrien, O. & Waugh, R. E. Cell adhesion molecule distribution relative to neutrophil surface topography assessed by TIRFM. Biophys. J. 97, 379–387 (2009)

    ADS  Article  PubMed  PubMed Central  Google Scholar 

  10. Moore, K. L. et al. P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin. J. Cell Biol. 128, 661–671 (1995)

    CAS  Article  PubMed  Google Scholar 

  11. Waugh, R. E. in Leukocyte Adhesion (ed. Ley, K. ) Ch. 1, 3–24 (Academic, 2009).

  12. Sundd, P. et al. Live cell imaging of paxillin in rolling neutrophils by dual-color quantitative dynamic footprinting. Microcirculation 18, 361–372 (2011)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Faust, N., Varas, F., Kelly, L. M., Heck, S. & Graf, T. Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood 96, 719–726 (2000)

    CAS  PubMed  Google Scholar 

  14. Pendl, G. G. et al. Immature mouse dendritic cells enter inflamed tissue, a process that requires E- and P-selectin, but not P-selectin glycoprotein ligand 1. Blood 99, 946–956 (2002)

    CAS  Article  PubMed  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  16. Xu, H. et al. Characterization of murine intercellular adhesion molecule-2. J. Immunol. 156, 4909–4914 (1996)

    CAS  PubMed  Google Scholar 

  17. Zarbock, A., Lowell, C. A. & Ley, K. Spleen tyrosine kinase Syk is necessary for E-selectin-induced αLβ2 integrin-mediated rolling on intercellular adhesion molecule-1. Immunity 26, 773–783 (2007)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Lawrence, M. B. & Springer, T. A. Leukocytes roll on a selectin at physiological flow rates: distinction from and prerequisite for adhesion through integrins. Cell 65, 859–873 (1991)

    CAS  Article  PubMed  Google Scholar 

  19. Kuwano, Y., Spelten, O., Zhang, H., Ley, K. & Zarbock, A. Rolling on E- or P-selectin induces the extended but not high-affinity conformation of LFA-1 in neutrophils. Blood 116, 617–624 (2010)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Woodfin, A. et al. The junctional adhesion molecule JAM-C regulates polarized transendothelial migration of neutrophils in vivo. Nature Immunol. 12, 761–769 (2011)

    CAS  Article  Google Scholar 

  21. Huang, M. T. et al. ICAM-2 mediates neutrophil transmigration in vivo: evidence for stimulus specificity and a role in PECAM-1-independent transmigration. Blood 107, 4721–4727 (2006)

    CAS  Article  PubMed  Google Scholar 

  22. Marshall, B. T. et al. Direct observation of catch bonds involving cell-adhesion molecules. Nature 423, 190–193 (2003)

    CAS  ADS  Article  PubMed  Google Scholar 

  23. Xia, L. et al. P-selectin glycoprotein ligand-1-deficient mice have impaired leukocyte tethering to E-selectin under flow. J. Clin. Invest. 109, 939–950 (2002)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Ding, Z. M. et al. Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration. J. Immunol. 163, 5029–5038 (1999)

    CAS  PubMed  Google Scholar 

  25. Hattori, R., Hamilton, K., Fugate, R., McEver, R. & Sims, P. Stimulated secretion of endothelial von Willebrand factor is accompanied by rapid redistribution to the cell surface of the intracellular granule membrane protein GMP-140. J. Biol. Chem. 264, 7768–7771 (1989)

    CAS  PubMed  Google Scholar 

  26. Doyle, E. L. et al. CD63 is an essential cofactor to leukocyte recruitment by endothelial P-selectin. Blood 118, 4265–4273 (2011)

    CAS  Article  PubMed  Google Scholar 

  27. Atarashi, K., Hirata, T., Matsumoto, M., Kanemitsu, N. & Miyasaka, M. Rolling of Th1 cells via P-selectin glycoprotein ligand-1 stimulates LFA-1-mediated cell binding to ICAM-1. J. Immunol. 174, 1424–1432 (2005)

    CAS  Article  PubMed  Google Scholar 

  28. Tojo, A. et al. Nitric oxide generated by nNOS in the macula densa regulates the afferent arteriolar diameter in rat kidney. Med. Electron Microsc. 37, 236–241 (2004)

    CAS  Article  PubMed  Google Scholar 

  29. Ley, K. et al. Sequential contribution of L- and P-selectin to leukocyte rolling in vivo. J. Exp. Med. 181, 669–675 (1995)

    CAS  Article  PubMed  Google Scholar 

  30. Kunkel, E. J. et al. Absence of trauma-induced leukocyte rolling in mice deficient in both P-selectin and intercellular adhesion molecule 1. J. Exp. Med. 183, 57–65 (1996)

    CAS  Article  PubMed  Google Scholar 

  31. Sperandio, M. et al. Severe impairment of leukocyte rolling in venules of core 2 glucosaminyltransferase-deficient mice. Blood 97, 3812–3819 (2001)

    CAS  Article  PubMed  Google Scholar 

  32. Schmid-Schönbein, G. W. Leukocyte biophysics. An invited review. Cell Biophys. 17, 107–135 (1990)

    Article  PubMed  Google Scholar 

  33. Pospieszalska, M. K. & Ley, K. in Leukocyte Adhesion (ed. Ley, K. ) Ch. 8 221–296 (Academic, 2009)

    Google Scholar 

  34. Brochard-Wyart, F., Borghi, N., Cuvelier, D. & Nassoy, P. Hydrodynamic narrowing of tubes extruded from cells. Proc. Natl Acad. Sci. USA 103, 7660–7663 (2006)

    CAS  ADS  Article  PubMed  PubMed Central  Google Scholar 

  35. Goldman, A. J., Cox, R. G. & Brenner, H. Slow viscous motion of a sphere parallel to a plane wall. II. Couette flow. Chem. Eng. Sci. 22, 653–660 (1967)

    CAS  Article  Google Scholar 

  36. Pospieszalska, M. K., Zarbock, A., Pickard, J. E. & Ley, K. Event-tracking model of adhesion identifies load-bearing bonds in rolling leukocytes. Microcirculation 16, 115–130 (2009)

    CAS  Article  PubMed  Google Scholar 

  37. Krasik, E. F. & Hammer, D. A. A semianalytic model of leukocyte rolling. Biophys. J. 87, 2919–2930 (2004)

    CAS  ADS  Article  PubMed  PubMed Central  Google Scholar 

  38. Goldman, A. J., Cox, R. G. & Brenner, H. Slow viscous motion of a sphere parallel to a plane wall. I. Motion through a quiescent fluid. Chem. Eng. Sci. 22, 637–651 (1967)

    CAS  Article  Google Scholar 

  39. Wilcox, R. R. New Statistical Procedures For The Social Sciences: Modern Solutions To Basic Problems (Lawrence Erlbaum Associates, 1987)

    Google Scholar 

Download references

Acknowledgements

The authors thank A. Zychlinsky for comments and reading the manuscript. This study was supported by the NCRP-Scientist Development Grant 11SDG7340005 from the American Heart Association (P.S.), WSA postdoctoral fellowship 10POST4160142-01 from American Heart Association (E.K.K.) and NIH EB 02185 (K.L.).

Author information

Authors and Affiliations

Authors

Contributions

P.S. performed all the experiments and image analysis. E.G. and A.G. designed the microfluidic device. M.K.P. calculated the fraction of bond force and torque shared by slings and tethers. E.K.K. was involved in culturing of Th1 CD4 T cells. Y.K. and S.F performed the scanning electron microscopy. P.S. and K.L. wrote the manuscript. K.L. supervised the project. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Klaus Ley.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-28, Supplementary Notes 1-4 and legends for Supplementary Movies 1-9. (PDF 9702 kb)

Supplementary Movie 1

Slings formed by DiI-stained mouse bone marrow neutrophil rolling on P-selectin - see Supplementary Information file for full legend. (MOV 1664 kb)

Supplementary Movie 2

Sling formed by an EGFP neutrophil rolling on P-selectin in whole blood of Lyz2-EGFP mouse - see Supplementary Information file for full legend. (MOV 918 kb)

Supplementary Movie 3

Wrapping of slings around a DiI-stained mouse bone marrow neutrophil rolling on P-selectin - see Supplementary Information file for full legend. (MOV 2828 kb)

Supplementary Movie 4

Wrapping of sling by a leukocyte rolling in the cremaster venule of a WT mouse - see Supplementary Information file for full legend. (MOV 216 kb)

Supplementary Movie 5

Sling formation by a leukocyte rolling in the cremaster venule of a WT mouse. Image processed to reveal sling - see Supplementary Information file for full legend. (MOV 75 kb)

Supplementary Movie 6

Tether (arrowhead) swings over to become a sling (arrow) - see Supplementary Information file for full legend. (MOV 222 kb)

Supplementary Movie 7

Tether (arrowhead) swings over to become a sling (arrow) - see Supplementary Information file for full legend. (MOV 1855 kb)

Supplementary Movie 8

Step-wise peeling of a sling. PSGL-1 patches (red spots) visible on sling (green) - see Supplementary Information file for full legend. (MOV 3116 kb)

Supplementary Movie 9

Step-wise peeling of a sling. PSGL-1 patches (red spots) visible on sling (green) - see Supplementary Information file for full legend. (MOV 2980 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sundd, P., Gutierrez, E., Koltsova, E. et al. ‘Slings’ enable neutrophil rolling at high shear. Nature 488, 399–403 (2012). https://doi.org/10.1038/nature11248

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature11248

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing