LPS stimulating neutrophils firmly adhered to ICAM-1 to form extracellular traps depends on integrin Mac-1 and cytoskeletal proteins

中性粒细胞胞外诱捕网（NETs）在免疫血栓的形成过程中扮演着重要角色，然而血管内皮细胞如何介导 NETs 的形成，尚未完全明晰。我们利用脂多糖（LPS）或佛波酯（PMA）对稳定黏附于内皮细胞表面细胞间黏附分子-1（ICAM-1）的中性粒细胞进行 4 h 刺激，再利用 Sytox green 染料标记 NETs-DNA，通过荧光显微镜观察 NETs 形成，分析 NETs-DNA 的面积和荧光强度以量化 NETs 的形成情况。结果显示，PMA 和 LPS 均能诱导稳定黏附于 ICAM-1 上的中性粒细胞产生 NETs。PMA 诱导产生的 NETs 不依赖于 β2 整合素淋巴细胞功能相关抗原-1（LFA-1）或巨噬细胞抗原复合物-1（Mac-1）。相反，LPS 刺激产生 NETs 依赖于 β2 整合素中 Mac-1，而不依赖 LFA-1。抑制肌动蛋白丝或踝蛋白-1（Talin-1），无论是 LPS 或 PMA 刺激，NETs 的形成均显著下降。本研究揭示了炎症条件下中性粒细胞与内皮细胞直接相互作用产生 NETs 的机制，为相关疾病的治疗和新药物的开发提供了新的理论基础。.; Neutrophil extracellular traps (NETs) play an important role in the formation of immunothrombosis. However, how vascular endothelial cells mediate the formation of NETs has not been fully understood. We stimulated neutrophils firmly attached on the endothelial cell surface intercellular adhesion molecule-1 (ICAM-1) with lipopolysaccharide (LPS) or phorbol-12-myristate-13-acetate (PMA) for 4 h, then labeled NETs-DNA with Sytox green dye and the formation of NETs was observed by fluorescent microscopy. The area and fluorescence intensity of NETs-DNA were analyzed to quantify the formation of NETs. The results showed that both PMA and LPS were able to induce firmly adhered neutrophils on ICAM-1 to produce NETs. NETs induced by PMA were independent of neither β2 integrin lymphocyte function-associated antigen-1 (LFA-1) nor macrophage antigen complex-1 (Mac-1). In contrast, LPS-stimulated NETs were mediated by Mac-1 integrin, but not by LFA-1. After inhibition of actin filaments or Talin-1, the formation of NETs irrespective of the stimulus was significantly reduced. This study reveals the mechanism of the direct interaction between neutrophils and endothelial cells to produce NETs under inflammatory conditions, providing a new theoretical basis for the treatment of related diseases and the development of new drugs.