Objective:To determine the protective effect of cardio-pulmonary bypass(CPB) with autologous lung as oxygenator on CPB-relative inflammatory response.Methods:Twelve adult mongrel dogs were randomly divided into control group and study group.Cardiopulmonary bypass (CPB) using a membrane oxygenator (control group) or using the autologous lung (study group) for gas exchange was performed for 120 min in an alternating series of 12 mongrel dogs with the heart arrested for 90 min by crystalloid cardioplegia and 30 min reperfusion.The blood samples were collected at the same time point of pre-operation(T1),60 min of cardiopulmonary bypass(T2),and 1 hour(T3),2 hours(T4) after cardiopulmonary bypass. Plasma concentration of IL-6,IL-10,TNF-α were detected with ELISA.Results:The plasma levels of IL-6,IL-8,IL-10,TNF-α in each group were significantly increased at T2,T3,T4.The plasma levels of IL-6,IL-8 and TNF-α in study group were significantly lower than in the control group at T2,T3,T4.The plasma levels of IL-10 in study group were significantly higher than the levels in control group at T2,T3,T4.Conclusion:This study indicates that extracorporeal circulation with autologous lung as oxygenator could reduce the increased amplitude of plasma levels of TNF-α,IL-6 and IL-8 whereas enhance the increased amplitude of the plasma IL-10 levels that result from CPB.In other word,extracorporeal circulation with autologous lung as oxygenator possesses the effect to regulate inflammatory cytokine balance and down-regulate CPB-relative inflammatory response.

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.
Cytoskeletal Proteins ; Endothelial Cells ; Extracellular Traps ; Integrins ; Intercellular Adhesion Molecule-1 ; Lipopolysaccharides/pharmacology* ; Macrophages ; Neutrophils

Extracellular traps released by neutrophils (neutrophil extracellular traps, NETs) are a double-edged sword, and understanding the mechanism of NET formation is of great significance for disease treatment. However, the short lifespan, the large individual differences, and the inability to perform gene editing render it difficult to decipher NET formation using neutrophils. It is necessary to find a model cell to replace neutrophils to study the mechanism of NET formation. In this study, we used different concentrations (0, 0.1, 1, and 10 μmol/L) of all-trans retinoic acid (ATRA) to differentiate HL-60 cells for different days (1, 3, 5, and 7 days). By detecting the cell viability and nuclear morphology of cells, we confirmed that HL-60 cells were differentiated to neutrophil-like cells (dHL-60) after treated with ATRA for at least 5 days. Using immunofluorescence staining to detect the formation of NETs, we demonstrated that dHL-60 cells differentiated for 5 days with 1 μmol/L ATRA could generate NETs comparable to those produced by neutrophils upon phorbol 12-myristate 13-acetate (PMA) stimulation, without histone H3 citrullination. Furthermore, the formation of NETs by dHL-60 cells were NADPH-dependent and PAD4-independent, consistent with neutrophils. Taken together, these observations suggest that dHL-60 cells differentiated with 1 μmol/L ATRA for 5 days can be used as a model cell for neutrophils to study the mechanism of NET formation.
Humans ; Extracellular Traps ; Tetradecanoylphorbol Acetate/pharmacology* ; Neutrophils ; HL-60 Cells ; Tretinoin/pharmacology*