Integrin-dependent cell adhesion to neutrophil extracellular traps through engagement of fibronectin in neutrophil-like cells

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 alpha 5 beta 1 and alpha v beta 3 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 alpha 5 beta 1 and alpha v beta 3 integrins, were subjected to adhesion assays in the presence or absence of DNAse 1, blocking antibodies against alpha 5 beta 1 or alpha v beta 3 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 alpha 5 beta 1 or alpha v beta 3 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 alpha 5 beta 1 and alpha v beta 3 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.