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

Abstract

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.

Synthesis of Human Neutrophil Extracellular Traps Contributes to Angiopoietin-Mediated In Vitro Proinflammatory and Proangiogenic Activities

“…In this article, we show that NETs, independent of the stimuli (e.g., angiopoietins or PMA), induce the binding of neutrophils onto hECM and HUVEC in a concentration-dependent manner. This effect appears to be dependent on NET integrity because their degradation with DNase I (4) completely abrogated neutrophil adhesion onto both hECM and ECs, which is in agreement with previous studies (61,62). In addition, the combination of DNase I with PMA alone (in the absence of NETs) did not alter the capacity of PMA to promote neutrophil adhesion onto hECM and HUVEC.…”

JAK2-V617F promotes venous thrombosis through β1/β2 integrin activation

“…Indeed in a recently published article, granulocytes were identified to play a pivotal role in venous thrombus formation in JAK2-V617F-knockin mice (66), supporting our finding that neutrophils are the major player in JAK2-V617F-driven thrombosis. The mechanism involved is increased formation of NETs (66,67). Thus, that finding and the results presented here point to a scenario wherein increased Rap1 activation induced by JAK2-V617F results in increased adhesion of granulocytes to the endothelium, which in turn releases NETs, thereby triggering increased thrombus formation.…”

Neutrophil accumulation and NET release contribute to thrombosis in HIT

“…The infusion of human plasma containing PF4 was also found to confer resistance to DNase I-mediated digestion (Supplemental Figure 5), and similar results were observed for digestion with bacterial-derived micrococcal nuclease (Supplemental Figure 6). Comparable nuclease resistance was also observed in a static system not coated with fibronectin, showing that fibronectin, which has a DNA-binding domain and is known to interact with NETs (27), is not the cause of the observed changes in NET behavior (Supplemental Figure 5).…”