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Context-Dependent Role of Vinculin in Neutrophil Adhesion, Motility and Trafficking.

ABSTRACT: Neutrophils are innate immune effector cells that traffic from the circulation to extravascular sites of inflammation. ?2 integrins are important mediators of the processes involved in neutrophil recruitment. Although neutrophils express the cytoskeletal protein vinculin, they do not form mature focal adhesions. Here, we characterize the role of vinculin in ?2 integrin-dependent neutrophil adhesion, migration, mechanosensing, and recruitment. We observe that knockout of vinculin attenuates, but does not completely abrogate, neutrophil adhesion, spreading, and crawling under static conditions. However, we also found that vinculin deficiency does not affect these behaviors in the presence of forces from fluid flow. In addition, we identify a role for vinculin in mechanosensing, as vinculin-deficient neutrophils exhibit attenuated spreading on stiff, but not soft, substrates. Consistent with these findings, we observe that in vivo neutrophil recruitment into the inflamed peritoneum of mice remains intact in the absence of vinculin. Together, these data suggest that while vinculin regulates some aspects of neutrophil adhesion and spreading, it may be dispensable for ?2 integrin-dependent neutrophil recruitment in vivo.

SUBMITTER: Wilson ZS

PROVIDER: S-EPMC7005776 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Context-Dependent Role of Vinculin in Neutrophil Adhesion, Motility and Trafficking.

Wilson Zachary S ZS Witt Hadley H Hazlett Lauren L Harman Michael M Neumann Brittany M BM Whitman Andrew A Patel Mohak M Ross Robert S RS Franck Christian C Reichner Jonathan S JS Lefort Craig T CT

Scientific reports 20200207 1

Neutrophils are innate immune effector cells that traffic from the circulation to extravascular sites of inflammation. β2 integrins are important mediators of the processes involved in neutrophil recruitment. Although neutrophils express the cytoskeletal protein vinculin, they do not form mature focal adhesions. Here, we characterize the role of vinculin in β2 integrin-dependent neutrophil adhesion, migration, mechanosensing, and recruitment. We observe that knockout of vinculin attenuates, but ...[more]

PMID: 32034208

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Project description:Neutrophil extracellular traps (NETs), originally recognized as a host defense mechanism, were reported to promote thrombosis and metastatic dissemination of cancer cells. Here we tested the role of integrins α5β1 and ανβ3 in the adhesion of cancer cells to NETs. Neutrophil-like cells stimulated with calcium ionophore (A23187) were used as a stable source of cell-free NETs-enriched suspensions. Using NETs as an adhesion substrate, two human K562 cell lines, differentially expressing α5β1 and ανβ3 integrins, were subjected to adhesion assays in the presence or absence of DNAse 1, blocking antibodies against α5β1 or ανβ3, alone or in combination with DNAse 1, and Proteinase K. As expected DNAse 1 treatment strongly inhibited adhesion of both cell lines to NETs. An equivalent significant reduction of cell adhesion to NETs was obtained after treatment of cells with blocking antibodies against α5β1 or ανβ3 indicating that both integrins were able to mediate cell adhesion to NETs. Furthermore, the combination of DNAse 1 and anti-integrin antibody treatment almost completely blocked cell adhesion. Western blot analysis and immunoprecipitation experiments showed a dose-dependent increase of fibronectin levels in samples from stimulated neutrophil-like cells and a direct or indirect interaction of fibronectin with histone H3. Finally, co-immunolocalization studies with confocal microscopy showed that fibronectin and citrullinated histone H3 co-localize inside the web-structure of NETs. In conclusion, our study showed that α5β1 and ανβ3 integrins mediate cell adhesion to NETs by binding to their common substrate fibronectin. Therefore, in addition to mechanical trapping and aspecific adsorption of different cell types driven by DNA/histone complexes, NETs may provide specific binding sites for integrin-mediated cell adhesion of neutrophils, platelets, endothelial and cancer cells thus promoting intimate interactions among these cells.

Dataset Information

Context-Dependent Role of Vinculin in Neutrophil Adhesion, Motility and Trafficking.

Publications

Context-Dependent Role of Vinculin in Neutrophil Adhesion, Motility and Trafficking.

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