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.
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。.

OBJECTIVE: PMNs (polymorphonuclear neutrophil) play important roles in early stage of inflammation induced ARDS (Acute Respiratory Distress Syndrome). Both HBP (Heparin-Binding Protein) released from active PMNs and β2 integrins on the surface of PMNs are involved in vascular leakage. The role and relationship of HBP and β2 integrins on ARDS still requires study.
METHODS: We established ARDS model using C57BL/6 mice with cecal ligation and puncture and eliminating HBP and β2 integrin with respective antibodies. The mice were also challenged with HBP endotracheal instillation. Histopathology score, lung wet/dry ratio, bronchoalveolar lavage fluid protein, plasma HBP and β2 integrin on PMNs from all groups were measured. β2 integrin and HBP were analyzed after incubated PMNs with streptococcal and pretreat with anti-CD18, anti-HBP, 1-phosphatidylinositol 3-kinase (PI3K) inhibitor and p38 mitogen-activated protein kinase (MAPK) inhibitor.
RESULTS: All lung injury indicatrix accompanied with HBP and β2 integrin elevated in CLP group, and HBP and β2 integrin were in correlation with each other and both were in correlation with the severity of lung injury. Endotracheal instillation HBP induced lung injury in CLP mice. Inhibiting both HBP and integrin ameliorated lung injury. HBP release was suppressed by inhibiting integrin and PI3K pathway, while integrin level did not decrease after eliminating HBP.
CONCLUSIONS: Both HBP and β2 integrin play important roles in ARDS. HBP released from PMNs is β2 integrin-PI3K signaling pathway dependent process revealing potential novel therapeutic targets for ARDS treatment.

The functional performance of the αI domain α7 helix in β2 integrin activation depends on the allostery of the α7 helix, which axially slides down; therefore, it is critical to elucidate what factors regulate the allostery. In this study, we determined that there were two conservative salt bridge interaction pairs that constrain both the upper and bottom ends of the α7 helix. Molecular dynamics (MD) simulations for three β2 integrin members, lymphocyte function-associated antigen-1 (LFA-1; αL β2 ), macrophage-1 antigen (Mac-1; αM β2 ) and αx β2 , indicated that the magnitude of the salt bridge interaction is related to the stability of the αI domain and the strength of the corresponding force-induced allostery. The disruption of the salt bridge interaction, especially with double mutations in both salt bridges, significantly reduced the force-induced allostery time for all three members. The effects of salt bridge interactions of the αI domain α7 helix on β2 integrin conformational stability and allostery were experimentally validated using Mac-1 constructs. The results demonstrated that salt bridge mutations did not alter the conformational state of Mac-1, but they did increase the force-induced ligand binding and shear resistance ability, which was consistent with MD simulations. This study offers new insight into the importance of salt bridge interaction constraints of the αI domain α7 helix and external force for β2 integrin function.

CD11b, the α-chain of β2 integrin Mac-1, is involved in many activation processes of phagocytes. Depending on the respective autoimmune disorder, CD11b has been shown to exert pro-inflammatory functions or be dispensable in their pathogenesis. Here, we investigated the role of CD11b in the pathogenesis of experimental epidermolysis bullosa acquisita (EBA), an autoimmune skin blistering disease mediated by autoantibodies to type VII collagen. Unexpectedly, in an antibody transfer-induced model of EBA, CD11b-deficient mice developed more severe disease symptoms than wild-type mice in the late phase of the disease. Furthermore, as compared to wild-type controls, CD11b-deficient mice expressed increased levels of circulating IFN-γ and IL-4. Taken together, for the first time, our results suggest an anti-inflammatory role for CD11b in experimental autoimmune diseases.