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‘Slings’ enable neutrophil rolling at high shear

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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.

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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.

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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.

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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)

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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

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