Integrin-dependent cell adhesion to neutrophil extracellular traps through engagement of fibronectin in neutrophil-like cells.
Integrin-dependent cell adhesion to neutrophil extracellular traps through engagement of fibronectin in neutrophil-like cells.
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DOI：
10.1371/journal.pone.0171362
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Del Vecchio S
中科院分区：
文献类型：
--
作者：
Monti M;Iommelli F;De Rosa V;Carriero MV;Miceli R;Camerlingo R;Di Minno G;Del Vecchio S
神经细胞外陷阱（NETs）最初被认为是一种宿主防御机制，据报道可促进血栓形成和癌细胞的转移扩散。在此，我们测试了整合素α5β1和ανβ3在癌细胞粘附于NET中的作用。用钙离子载体（A23187）刺激的嗜中性粒细胞样细胞用作无细胞NET富集悬浮液的稳定来源。使用NET作为粘附底物，在存在或不存在DNA酶1、针对α 5 β 1或α ν β3的阻断抗体（单独或与DNA酶1组合）和蛋白酶K的情况下，对差异表达α 5 β 1和α ν β3整联蛋白的两种人K562细胞系进行粘附测定。正如预期的那样，DNA酶1处理强烈抑制了两种细胞系与NET的粘附。在用针对α5β1或ανβ3的阻断抗体处理细胞后，获得了与NET的细胞粘附的同等显著降低，表明两种整联蛋白都能够介导与NET的细胞粘附。此外，DNA酶1和抗整联蛋白抗体处理的组合几乎完全阻断细胞粘附。Western印迹分析和免疫沉淀实验表明，从刺激的嗜中性粒细胞样细胞和直接或间接的相互作用的纤连蛋白与组蛋白H3的样品中的纤连蛋白水平的剂量依赖性增加。最后，共聚焦显微镜的共免疫定位研究表明，纤连蛋白和瓜氨酸化组蛋白H3共定位在NET的网络结构内。总之，我们的研究表明，α5β1和ανβ3整合素通过与它们共同的底物纤连蛋白结合来介导细胞与NET的粘附。因此，除了由DNA/组蛋白复合物驱动的不同细胞类型的机械捕获和非特异性吸附之外，NET可以为中性粒细胞、血小板、内皮细胞和癌细胞的整合素介导的细胞粘附提供特异性结合位点，从而促进这些细胞之间的密切相互作用。
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.