Monti Marcello, Iommelli Francesca, De Rosa Viviana, Carriero Maria Vincenza, Miceli Roberta, Camerlingo Rosa, Di Minno Giovanni, Del Vecchio Silvana
Dipartimento di Medicina Clinica e Chirurgia, Università degli Studi di Napoli "Federico II", Naples, Italy.
Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche, Naples, Italy.
PLoS One. 2017 Feb 6;12(2):e0171362. doi: 10.1371/journal.pone.0171362. eCollection 2017.
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.
中性粒细胞胞外陷阱（NETs）最初被认为是一种宿主防御机制，据报道它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在癌细胞与NETs黏附中的作用。用钙离子载体（A23187）刺激的类中性粒细胞用作富含无细胞NETs的稳定悬浮液来源。以NETs作为黏附底物，对两种差异表达α5β1和ανβ3整合素的人K562细胞系进行黏附试验，试验条件包括存在或不存在DNA酶1、单独或与DNA酶1联合使用的针对α5β1或ανβ3的阻断抗体以及蛋白酶K。正如预期的那样，DNA酶1处理强烈抑制了两种细胞系与NETs的黏附。用针对α5β1或ανβ3的阻断抗体处理细胞后，细胞与NETs的黏附也显著同等程度降低，这表明这两种整合素都能够介导细胞与NETs的黏附。此外，DNA酶1和抗整合素抗体联合处理几乎完全阻断了细胞黏附。蛋白质印迹分析和免疫沉淀实验表明，来自受刺激的类中性粒细胞的样品中纤连蛋白水平呈剂量依赖性增加，且纤连蛋白与组蛋白H3存在直接或间接相互作用。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在NETs的网状结构内共定位。总之，我们的研究表明，α5β1和ανβ3整合素通过与其共同底物纤连蛋白结合来介导细胞与NETs的黏附。因此，除了由DNA/组蛋白复合物驱动的对不同细胞类型的机械捕获和非特异性吸附外，NETs可能为中性粒细胞、血小板、内皮细胞和癌细胞的整合素介导的细胞黏附提供特定的结合位点，从而促进这些细胞之间的密切相互作用。
