| Neutrophil extracellular traps(NETs)play an important role in regulating the formation of immunethrombosis.Regarding the formation of intravascular NETs under infection conditions,most of the current research focuses on the mechanism of activated platelets-mediated the formation of NETs.However,there are relatively few studies on the formation of NETs mediated by endothelial cells.Under infected conditions,vascular endothelial cells significantly express ICAM-1 molecules,and it is not fully understood whether the direct interaction between neutrophils and vascular endothelial cells ICAM-1 can cause the production of NETs and the mechanisms mediating the formation of NETs in this process.We stimulated neutrophils stably attached on the endothelial cell surface molecule ICAM-1 with lipopolysaccharide LPS or PMA for 4 h,then labeled NETs-DNA with Sytox green dye and labeled the specific biomarker of NETs formation,Cit H3.The formation of NETs were observed by fluorescent microscopy and analyzed to quantify the formation of NETs by Image J.The results showed that both PMA and LPS were able to induce stable adhered neutrophils on ICAM-1 to produce NETs.NETs induced by PMA were independent of neither β2 integrin LFA-1 nor 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.To investigate intracellular signaling pathways,knocking out the relevant protein genes is a direct and effective approach.However,because neutrophils are terminally differentiated cells and have a short lifespan,they cannot be genetically manipulated to investigate signaling pathways.HL-60 cells are human promyelocytic leukemia cells that are often used to induce differentiation into neutrophil-like differentiation of HL-60(d HL-60)cells to study the biological functions of neutrophils.HL-60 cells serve as model cells that can be genetically manipulated and are ideal tools for the study of NETs formation signaling pathways.However,the optimal conditions for all-trans retinoic acid(ATRA)to be used as an inducer to differentiate HL-60 cells to form NETs are not fully understood at present.Therefore,in this paper,gradient concentrations of ATRA were set up for differentiation of HL-60 cells and treated for different days to explore the optimal conditions under which d HL-60 cells can produce NETs.The degree of HL-60 differentiation was examined by staining the nuclear morphology of differentiated HL-60 cells with Giemsa staining and by flow detection of LFA-1 and Mac-1 expression on HL-60 cells.The results showed that the degree of differentiation of HL-60 cells was time-dependent,and more than 80% of HL-60 cells successfully differentiated into bands cells or mature granulocytes after 7 days of differentiation;the expression of Mac-1 on HL-60 cells was significantly increased under 1μM as well as 10 μM ATRA induction.The results of PMA stimulation of NETs formation in ATRA-induced differentiation of d HL-60 cells showed that after 5 days of ATRA differentiation,NETs formation was strongest in HL-60 cells treated at 1 μM and 10 μM concentrations,significantly higher than that of HL-60 cells induced by the same differentiation days,and other ATRA concentrations.Also,it was significantly higher than the NETs formation of HL-60 cells at the same ATRA differentiation concentration,and other days of differentiation.We therefore recommend using ATRA at concentrations of 1 μM or 10μM to induce differentiation of HL-60 cells up to day 5 for NETs-related experiments.Based on the above findings,this study reveals the mechanism of the direct interaction between neutrophils and endothelial cells to produce NETs under inflammatory conditions as well as provides certain guidance and reference for researchers to use d HL-60 cells for NETs research,providing a new theoretical basis for the treatment of related diseases and the development of new drugs. |
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