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Cell and Tissue Research

, Volume 355, Issue 2, pp 375–396 | Cite as

Angiostatin inhibits activation and migration of neutrophils

  • Gurpreet K. Aulakh
  • Yadu Balachandran
  • Lixin Liu
  • Baljit SinghEmail author
Regular Article

Abstract

There is a critical need to identify molecules that modulate the biology of neutrophils because activated neutrophils, though necessary for host defense, cause exuberant tissue damage through production of reactive oxygen species and increased lifespan. Angiostatin, an endogenous anti-angiogenic cleavage product of plasminogen, binds to integrin αvβ3, ATP synthase and angiomotin and its expression is increased in inflammatory conditions. We test the hypothesis that angiostatin inhibits neutrophil activation, induces apoptosis and blocks recruitment in vivo and in vitro. The data show immuno-reactivity for plasminogen/angiostatin in resting neutrophils. Angiostatin conjugated to FITC revealed that angiostatin was endocytozed by activated mouse and human neutrophils in a lipid raft-dependent fashion. Co-immunoprecipitation of human neutrophil lysates, confocal microscopy of isolated mouse and human neutrophils and functional blocking experiments showed that angiostatin complexes with flotillin-1 along with integrin αvβ3 and ATP synthase. Angiostatin inhibited fMLP-induced neutrophil polarization, as well as caused inhibition of hsp-27 phosphorylation and stabilization of microtubules. Angiostatin treatment, before or after LPS-induced neutrophil activation, inhibited phosphorylation of p38 and p44/42 MAPKs, abolished reactive oxygen species production and released the neutrophils from suppressed apoptosis, as indicated by expression of activated caspase-3 and morphological evidence of apoptosis. Finally, intravital microscopy and myeloperoxidase assay showed inhibition of neutrophil recruitment in post-capillary venules of TNFα-treated cremaster muscle in mouse. These in vitro and in vivo data demonstrate angiostatin as a broad deactivator and silencer of neutrophils and an inhibitor of their migration. These data potentially open new avenues for the development of anti-inflammatory drugs.

Keywords

Neutrophil polarization Apoptosis Lipid raft Integrin αvβ3 Intravital microscopy 

Notes

Acknowledgments

We would like to thank Dr. Wolfgang Kuebler for providing valuable comments on the manuscript and Mr. Daryoush Hajinezhad for technical assistance with the confocal microscope. The study was funded through a grant from the Natural Sciences and Engineering Research Council of Canada to Dr. Baljit Singh. Dr. Aulakh was supported through a Dean’s Scholarship from the College of Graduate Studies and Research, University of Saskatchewan.

Authorship

G.K.A., L.L. and B.S. planned the experiments, analyzed data and wrote the manuscript. G.K.A. carried out all the experiments. Y.B. helped in angiostatin post-treatment confocal experiments on human neutrophils.

Supplementary material

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Gurpreet K. Aulakh
    • 1
  • Yadu Balachandran
    • 1
  • Lixin Liu
    • 2
  • Baljit Singh
    • 1
    Email author
  1. 1.Veterinary Biomedical Sciences, Western College of Veterinary MedicineUniversity of SaskatchewanSaskatoonCanada
  2. 2.Department of Pharmacology, College of MedicineUniversity of SaskatchewanSaskatoonCanada

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