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Kindlin-3 is required for β2 integrin–mediated leukocyte adhesion to endothelial cells

Abstract

Integrin activation is essential for the function of all blood cells, including platelets and leukocytes1. The blood cell–specific FERM domain protein Kindlin-3 is required for the activation of the β1 and β3 integrins on platelets2. Impaired activation of β1, β2 and β3 integrins on platelets and leukocytes is the hallmark of a rare autosomal recessive leukocyte adhesion deficiency syndrome in humans called LAD-III, characterized by severe bleeding and impaired adhesion of leukocytes to inflamed endothelia3. Here we show that Kindlin-3 also binds the β2 integrin cytoplasmic domain and is essential for neutrophil binding and spreading on β2 integrin-dependent ligands such as intercellular adhesion molecule-1 and the complement C3 activation product iC3b. Moreover, loss of Kindlin-3 expression abolished firm adhesion and arrest of neutrophils on activated endothelial cells in vitro and in vivo, whereas selectin-mediated rolling was unaffected. Thus, Kindlin-3 is essential to activate the β1, β2 and β3 integrin classes, and loss of Kindlin-3 function is sufficient to cause a LAD-III–like phenotype in mice.

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Figure 1: Characterization of PMNs in Kindlin3−/− chimeric mice.
Figure 2: Functional properties of Kindlin3−/− PMNs.
Figure 3: Abrogated leukocyte adhesion and extravasation in Kindlin3−/− chimeras.
Figure 4: Leukocyte rolling and adhesion under in vivo and in vitro conditions.

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Acknowledgements

We thank the members of the Fässler lab for discussions, R. Zent for careful reading of the manuscript, K. Kessenbrock for help with the oxidative burst experiment, T. Lämmermann for help with the skin inflammation assay and S. Dietzel for help with generating the movie clips. The work was funded by the Deutsche Forschungsgemeinschaft and the Max Planck Society.

Author information

Authors and Affiliations

Authors

Contributions

M.M., M.S. and R.F. designed and supervised research, analyzed data and wrote the paper. M.M., M.B., S. Schmid, R.R., S. Schmidt, M. Sixt, H.-V.W. and M. Sperandio performed experiments. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Reinhard Fässler.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–3, Supplementary Tables 1 and 2 and Supplementary Methods (PDF 252 kb)

Supplementary Video 1

Cell spreading on immobilized immune complexes. Spreading of Kindlin3+/+ and Kindlin3−/− PMNs was observed by differential interference contrast microscopy. (MOV 891 kb)

Supplementary Video 2

Leukocyte rolling and adhesion in control fetal liver cell chimeras. Intravital transillumination microscopy was used to visualize leukocyte rolling and adhesion in TNF-α-stimulated cremaster muscle venules of Kindlin3+/+ chimeric mice. Note the abundance of leukocytes firmly adherent on the inflamed endothelium and the rather low number of rolling leukocytes. (MOV 1546 kb)

Supplementary Video 3

Leukocyte rolling and adhesion in Kindlin3-/- fetal liver cell chimeras. Leukocyte rolling and adhesion in TNF-α-stimulated cremaster muscle venules of Kindlin3−/− mice was observed by intravital microscopy. Due to the severe adhesion defect in Kindlin3−/− mice, leukocyte adhesion is rare while many rolling leukocytes are seen. (MOV 1169 kb)

Supplementary Video 4

Leukocyte adhesion in Kindlin3+/+ fetal liver cell chimeras after blocking leukocyte rolling. Leukocyte adhesion was recorded in TNF-α-stimulated cremaster muscle venules of Kindlin3+/+ chimeric mice five minutes after injection of anti-E-selectin blocking mAb 9A9 and anti-P-selectin blocking mAb RB40.34. Leukocyte rolling is completely absent, while a significant number of leukocytes is still firmly adherent on the inflamed endothelium. (MOV 2035 kb)

Supplementary Video 5

Leukocyte adhesion in Kindlin3−/− fetal liver cell chimeras after blocking leukocyte rolling. Five minutes after systemic injection of anti-E-selectin blocking mAb 9A9 and anti-P-selectin blocking mAb RB40.34 into Kindlin3−/− chimeric mice, leukocyte rolling was completely blocked in TNF-α-stimulated cremaster muscle venules. In contrast to Kindlin3+/+ chimeric mice, leukocyte adhesion is almost completely absent in Kindlin3−/− chimeras illustrating the crucial role of Kindlin-3 for firm leukocyte adhesion. (MOV 855 kb)

Supplementary Video 6

Chemokine-induced leukocyte arrest in Kindlin3+/+ fetal liver cell chimeras following systemic injection of CXCL1. Leukocyte adhesion and rolling was recorded before, during, and after injection of 600ng CXCL1. Application of CXCL1 (indicated by the appearance of a white square) into Kindlin3+/+ mice led to a significant increase in leukocyte adhesion accompanied by a dramatic drop in leukocyte rolling. (MOV 4363 kb)

Supplementary Video 7

Chemokine-induced leukocyte arrest in Kindlin3−/− fetal liver cell chimeras following systemic injection of CXCL1. In contrast to Kindlin3+/+ mice, leukocyte adhesion and rolling in Kindlin3−/− mice were similar before, during (indicated by the white square), and after injection of 600ng CXCL1 demonstrating that Kindlin-3 is essential in mediating chemokine-triggered firm leukocyte arrest in vivo. (MOV 4224 kb)

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Moser, M., Bauer, M., Schmid, S. et al. Kindlin-3 is required for β2 integrin–mediated leukocyte adhesion to endothelial cells. Nat Med 15, 300–305 (2009). https://doi.org/10.1038/nm.1921

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