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Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury

Abstract

Selectins and their ligands mediate leukocyte rolling, allowing interactions with chemokines that lead to integrin activation and arrest. Here we show that E-selectin is crucial for generating a secondary wave of activating signals, transduced specifically by E-selectin ligand-1, that induces polarized, activated αMβ2 integrin clusters at the leading edge of crawling neutrophils, allowing capture of circulating erythrocytes or platelets. In a humanized mouse model of sickle cell disease, the capture of erythrocytes by αMβ2 microdomains leads to acute lethal vascular occlusions. In a model of transfusion-related acute lung injury, polarized neutrophils capture circulating platelets, resulting in the generation of oxidative species that produce vascular damage and lung injury. Inactivation of E-selectin or αMβ2 prevents tissue injury in both inflammatory models, suggesting broad implications of this paradigm in thromboinflammatory diseases. These results indicate that endothelial selectins can influence neutrophil behavior beyond its canonical rolling step through delayed, organ-damaging, polarized activation.

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Figure 1: Heterotypic interactions of RBCs and platelets with leukocyte microdomains are induced during inflammation.
Figure 2: RBC and platelet interactions with leukocytes depend on E-selectin and its ligand ESL-1.
Figure 3: Heterotypic interactions with RBCs and platelets are mediated by the leukocyte integrin αMβ2.
Figure 4: E-selectin and ESL-1 modulate regional αMβ2 activity on adherent leukocytes in cremasteric venules in vivo.
Figure 5: Antibody-induced lung injury requires platelet-leukocyte interactions and is blocked by antibodies to E-selectin and αMβ2.
Figure 6: Vaso-occlusion in sickle cell disease requires E-selectin–mediated activation of αMβ2 integrin.

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Acknowledgements

We thank C. Jakubzick for help establishing the lung injury model and B. Wolitzky (Immune Tolerance Network) for the 9A9 antibody to E-selectin. This work was supported by US National Institutes of Health grants R01 HL69438 to P.S.F. and T32 HL07824 to J.C. and by a Scientist Development Grant from the American Heart Association to A.H. (0735165N). P.S.F. is supported by an Established Investigator Award from the American Heart Association. A.H. is the recipient of a Ramón y Cajal fellowship from the Spanish Ministry of Science and Innovation.

Author information

Authors and Affiliations

Authors

Contributions

A.H. conceived the study, performed experiments, analyzed data and wrote the manuscript; J.C. performed experiments and analyzed data; A.J.P. maintained and generated the mice used in this study; J.-E.J. performed experiments and analyzed data; E.Y.C. performed experiments and analyzed data; and P.S.F. conceived the study, supervised the overall project, analyzed data and wrote the manuscript.

Corresponding authors

Correspondence to Andrés Hidalgo or Paul S Frenette.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–11, Supplementary Tables 1 and 2 and Supplementary Methods (PDF 543 kb)

Supplementary Video 1

nRBC captures are mediated by the leading edge of adherent PMNs. Adherent leukocytes in venules of C57BL/6 mice treated with TNF-α were imaged following the intravenous injection of PE-conjugated anti-L-selectin (red, 0.5 μg) and FITC-conjugated anti-LFA-1 (clone M17/4; green, 1 μg). L-selectin clusters identify the trailing edge of adherent leukocytes. Brightfield images of nRBC interactions with PMNs in inflamed venules were captured 180 min after cytokine administration. (MOV 1362 kb)

Supplementary Video 2

Platelets interact mostly with leukocyte microdomains at the leading edge. Platelets were labeled by anti-CD41 (red, 1 μg / mouse) and the trailing edge with anti-L-selectin (blue, 0.02 mg/Kg) in a TNF-α-stimulated mouse. Real time is shown in the left upper corner (h:min:s). (MOV 1643 kb)

Supplementary Video 3

Regional activation of αMβ2 integrin at the leading edge of adherent leukocytes mediates fluosphere capture. TNF-α-treated C57BL/6 mice were injected with PE-conjugated anti-L-selectin (red; 0.5 μg) to label the trailing edge of adherent leukocytes. Fluosphere interactions with adherent leukocytes in inflamed venules were imaged 180 min after cytokine treatment, immediately upon the injection of albumin-coated fluospheres (green) through a catheter placed in the left carotid artery. (MOV 7932 kb)

Supplementary Video 4a

Platelet-WBC interactions are markedly induced by anti-H2d administration in Balb/c mice. Platelets were labeled by anti-CD41 (red, 1 μg / mouse) and the trailing edge with anti-L-selectin (blue, 0.02 mg/Kg). (4a) Sequence of images just before anti-H2d administration. (MOV 4785 kb)

Supplementary Video 4b

Platelet-WBC interactions are markedly induced by anti-H2d administration in Balb/c mice. Platelets were labeled by anti-CD41 (red, 1 μg / mouse) and the trailing edge with anti-L-selectin (blue, 0.02 mg/Kg). (4b) Sequence of images taken after anti-H2d injection. Note the increase in platelet captures by leukocytes and also the interactions of non-labeled RBCs with adherent leukocytes. (MOV 4665 kb)

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Hidalgo, A., Chang, J., Jang, JE. et al. Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury. Nat Med 15, 384–391 (2009). https://doi.org/10.1038/nm.1939

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