The effect of neutrophil extracellular traps (NETs) on promoting intravascular microthrombi formation and exacerbating the severity of sepsis in patients has gained extensive attention. However, in sepsis, the mechanisms and key signaling molecules mediating NET formation during direct interactions of endothelial cells and neutrophils still need further explored. Herein, we utilized lipoteichoic acid (LTA), a component shared by Gram-positive bacteria, to induce NET extrusion from neutrophils firmly adhered to the glass slides coated with intercellular adhesion molecule-1(ICAM-1). We also used Sytox green to label NET-DNA and Flou-4 AM as the intracellular Ca2+ signaling indicator to observe the NET formation and fluctuation of Ca2+ signaling. Our results illustrated that LTA was able to induce NET release from neutrophils firmly attached to ICAM-1-coated glass slides, and the process was time-dependent. In addition, our study indicated that LTA-induced NET release by neutrophils stably adhered to ICAM-1 depended on Ca2+ signaling but not intracellular reactive oxygen species (ROS). This study reveals NET formation mediated by direct interactions between endothelial ICAM-1 and neutrophils under LTA stimulation and key signaling molecules involved, providing the theoretical basis for medicine development and clinical treatment for related diseases.
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.