LPS stimulating neutrophils firmly adhered to ICAM-1 to form extracellular traps depends on integrin Mac-1 and cytoskeletal proteins / 生物医学工程学杂志

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.

The role of integrin involved heparin-binding protein secretion in acute lung injury / 中华急诊医学杂志

Objective To study the relationship between heparin-binding protein(HBP) and β2 integrin on the surface of neutrophil in acute lung injury.Methods A total of 30 mice from animal center of Tongji University were divided into control group,CLP group and antibody-treatment group by random number method.Acute lung injury animal model was established by cecal Ligation and puncture in the mice of CLP group and antibody-treatment group.Sham operations were performed on the mice in control group.Mice in antibody-treatment group received anti-CD18 antibody injected via tail vein 30 min before establishing acute lung injury animal model.Twenty-four hours after operation,mice were sacrificed and lung tissue was taken.After HE staining,lung injury were evaluated by Smith score.Lung tissue wet/dry weight ratio,BALF protein level,plasma HBP level and β2 integrin on neutrophil were measured.T test were used to compare the difference among groups.Pearson correlation was used to study the correlation between HBP,integrin and lung injury index.Results Compared with control group,mice in CLP group had higher Smith score(t=10.607,P＜0.01),lung wet/dry ratio(t=3.968,P=0.001),BALF protein level(t=4.331,P＜0.01) and as well as higher plasma HBP(t=3.515,P=0.002) and β2 integrin(t=4.816,P＜0.01) level.After CD18 antibody treatment,anti-treat group mice had lower Smith score (t=2.307,P=0.033),lung wet/dry ratio(t=3.080,P=0.006),BALF protein level(t=2.484,P=0.023) and as well as higher plasma HBP level(t=2.218,P=0.046) than mice in CLP group.Pearson correlation analysis showed HBP had obvious correlation with wet/dry ratio(r=0.527,P=0.017),BALF protein(r=0.508,P=0.022) and as well as β2 integrin(r=0.674,P=0.001).Conclusions Both HBP and β2 integrin involved in the lung injury pathogenesis.HBP level is interrelated with the severity of lung injury.The β2 integrin is associated with the release of HBP by neutrophil.Inhabiting the function of integrin could decrease HBP level and alleviate the severity of lung injury.

Spreading dynamics of β2 integrin expressed human neutrophils onto ICAM-1-immobilized substrate / 医用生物力学

Objective To elucidate the spreading dynamics of β2 integrin expressed human neutrophils (PMNs) on ICAM-1-immobilized substrate. Methods The fraction of PMN spreading on the substrate pre coated by 10, 20, or 100 μg/mL intercellular adhesive molecule-1 (ICAM-1) was quantified when that on 2% human serum albumin (HSA) immobilized or that on blank substrate was served as control. The site density of β2 integrin expressing on PMNs was determined using flow cytometry and the regulation of β2 integrin subunits was defined using the fraction of PMN spreading on 100 μg/mL ICAM-1 substrate by blocking CD11a or CD11b subunit of β2 integrin. Results PMN spreading was presented on ICAM-1-immobilized substrate but absent on 2% HSA-immobilized substrate, supporting the specificity of β2 integrin induced spreading. Time course of neutrophil spreading on ICAM-1 substrate was density dependent of both ICAM-1 and β2 integrin molecules. The fraction of PMN spreading was reduced significantly when the expression of CD11b subunit was blocked. Conclusions PMN spreading was mediated specifically by β2 integrin-ICAM-1 interactions and determined by the expression of β2 integrin and ICAM-1, in which CD11b subunit played a dominate role.

RO-Heparin Inhibits β2-Integrin(Mac-1)-mediated Neutrophils Adhesion / 生物化学与生物物理进展

The adhesion of leukocytes to vascular endothelium is crucial for the generation of inflammatory responses. The selectinsand β2-integrin (Mac-1) play a major role in the process. Recently, it was reported that RO-heparin can inhibit selectin-mediated leukocyte adhesion. The effect of RO-heparin on the Mac-1-mediated neutrophils adhesion were further tested. The results showed that RO-heparin could effectively inhibit neutrophils binding to ICAM-1, adhering to COS-7 cells expressing human ICAM-1, and adheringto human umbilical vein endothelial cells (HUVECs) under static and flow conditions. The findings suggest that the effect of RO-heparin on leukocyte adhesion is mainly due to its inhibition on the interaction between selectins or Mac-1 and their ligands and that RO-heparin might be useful in preventing inflammation diseases.