1. Immunology and Inflammation
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Endothelial cell-derived CD95 ligand serves as a chemokine in induction of neutrophil slow rolling and adhesion

  1. Liang Gao
  2. Gülce Sila Gülcüler
  3. Lieke Golbach
  4. Helena Block
  5. Alexander Zarbock
  6. Ana Martin-Villalba  Is a corresponding author
  1. German Cancer Research Center, Germany
  2. University of Münster, Germany
Research Article
  • Cited 16
  • Views 1,266
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Cite this article as: eLife 2016;5:e18542 doi: 10.7554/eLife.18542

Abstract

Integrin activation is crucial for regulation of leukocyte rolling, adhesion and trans-vessel migration during inflammation and occurs by engagement of myeloid cells through factors presented by inflamed vessels. However, endothelial-dependent mechanisms of myeloid cell recruitment are not fully understood. Here we show using an autoperfused flow chamber assay of whole blood neutrophils and intravital microscopy of the inflamed cremaster muscle that CD95 mediates leukocyte slow rolling, adhesion and transmigration upon binding of CD95-ligand (CD95L) that is presented by endothelial cells. In myeloid cells, CD95 triggers activation of Syk-Btk/PLCγ2/Rap1 signaling that ultimately leads to integrin activation. Excitingly, CD95-deficient myeloid cells exhibit impaired bacterial clearance in an animal model of sepsis induced by cecal ligation and puncture (CLP). Our data identify the cellular and molecular mechanisms underlying the chemoattractant effect of endothelial cell-derived CD95L in induction of neutrophil recruitment and support the use of therapeutic inhibition of CD95's activity in inflammatory diseases.

Article and author information

Author details

  1. Liang Gao

    Division of Molecular Neurobiology, German Cancer Research Center, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Gülce Sila Gülcüler

    Division of Molecular Neurobiology, German Cancer Research Center, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Lieke Golbach

    Department of Anesthesiology and Critical Care Medicine, University of Münster, Münster, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Helena Block

    Department of Anesthesiology and Critical Care Medicine, University of Münster, Münster, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Alexander Zarbock

    Department of Anesthesiology and Critical Care Medicine, University of Münster, Münster, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Ana Martin-Villalba

    Division of Molecular Neurobiology, German Cancer Research Center, Heidelberg, Germany
    For correspondence
    a.martin-villalba@dkfz.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9405-8910

Funding

Deutsches Krebsforschungszentrum

  • Ana Martin-Villalba

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: Animal experiments in this study were performed in accordance with institutional guidelines of the German Cancer Research Center and were approved by the Regierungspräsidium Karlsruhe, Germany (Permit Number G188/13).

Reviewing Editor

  1. William Muller

Publication history

  1. Received: June 7, 2016
  2. Accepted: October 19, 2016
  3. Accepted Manuscript published: October 20, 2016 (version 1)
  4. Version of Record published: November 7, 2016 (version 2)

Copyright

© 2016, Gao et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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Further reading

    1. Immunology and Inflammation
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    Background: Control of the COVID-19 pandemic will rely on SARS-CoV-2 vaccine-elicited antibodies to protect against emerging and future variants; an understanding of the unique features of the humoral responses to infection and vaccination, including different vaccine platforms, is needed to achieve this goal.

    Methods: The epitopes and pathways of escape for Spike-specific antibodies in individuals with diverse infection and vaccination history were profiled using Phage-DMS. Principal component analysis was performed to identify regions of antibody binding along the Spike protein that differentiate the samples from one another. Within these epitope regions we determined potential sites of escape by comparing antibody binding of peptides containing wildtype residues versus peptides containing a mutant residue.

    Results: Individuals with mild infection had antibodies that bound to epitopes in the S2 subunit within the fusion peptide and heptad-repeat regions, whereas vaccinated individuals had antibodies that additionally bound to epitopes in the N- and C-terminal domains of the S1 subunit, a pattern that was also observed in individuals with severe disease due to infection. Epitope binding appeared to change over time after vaccination, but other covariates such as mRNA vaccine dose, mRNA vaccine type, and age did not affect antibody binding to these epitopes. Vaccination induced a relatively uniform escape profile across individuals for some epitopes, whereas there was much more variation in escape pathways in mildly infected individuals. In the case of antibodies targeting the fusion peptide region, which was a common response to both infection and vaccination, the escape profile after infection was not altered by subsequent vaccination.

    Conclusions: The finding that SARS-CoV-2 mRNA vaccination resulted in binding to additional epitopes beyond what was seen after infection suggests protection could vary depending on the route of exposure to Spike antigen. The relatively conserved escape pathways to vaccine-induced antibodies relative to infection-induced antibodies suggests that if escape variants emerge, they may be readily selected for across vaccinated individuals. Given that the majority of people will be first exposed to Spike via vaccination and not infection, this work has implications for predicting the selection of immune escape variants at a population level.

    Funding: This work was supported by NIH grants AI138709 (PI Overbaugh) and AI146028 (PI Matsen). Julie Overbaugh received support as the Endowed Chair for Graduate Education (FHCRC). The research of Frederick Matsen was supported in part by a Faculty Scholar grant from the Howard Hughes Medical Institute and the Simons Foundation. Scientific Computing Infrastructure at Fred Hutch was funded by ORIP grant S10OD028685.

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    T cell activation requires engagement of a cognate antigen by the T cell receptor (TCR) and the co-stimulatory signal of CD28. Both TCR and CD28 aggregate into clusters at the plasma membrane of activated T cells. While the role of TCR clustering in T cell activation has been extensively investigated, little is known about how CD28 clustering contributes to CD28 signalling. Here, we report that upon CD28 triggering, the BAR-domain protein sorting nexin 9 (SNX9) is recruited to CD28 clusters at the immunological synapse. Using three-dimensional correlative light and electron microscopy, we show that SNX9 generates membrane tubulation out of CD28 clusters. Our data further reveal that CD28 clusters are in fact dynamic structures and that SNX9 regulates their stability as well as CD28 phosphorylation and the resulting production of the cytokine IL-2. In summary, our work suggests a model in which SNX9-mediated tubulation generates a membrane environment that promotes CD28 triggering and downstream signalling events.