ROS promotes adhesion of neutrophils to bone marrow stromal cells and its mechanism

AIM:To investigate the effect of reactive oxygen species (ROS) on the adhesion of neutrophils to bone marrow stromal cells (BMSCs) and its mechanism. METHODS:Murine bone marrow neutrophils were isolated from mouse tibia and femur by density gradient centrifugation. HL60 cells were induced into human mature neutrophils (dHL60 cells) by DMSO treatment. Murine bone marrow neutrophils and dHL60 cells were labeled with CFDA-SE. The adhesion of the CFDA-SE-labeled cells to BMSC monolayer was tested by microplate reader after H₂O₂ treatment. The level of glutaredoxin 1 (Grx1) in dHL60 cells infected by lentivirus carrying Grx1 expression vector was examined by fluorescence microscopy and Western blot. The genotype of Grx1^-/- mice was identified by PCR. RESULTS:Diff-Quick staining result displayed that the purity of murine bone marrow neutrophil was higher than 90%. The adhesion of H₂O₂-pretreated neutrophils to BMSCs was higher than that of the control cells (P<0.01). The expression of Grx1 in Grx1 stably transfected dHL60 cells was significantly higher than that in the control cells. After H₂O₂ treatment, the results of in vitro adhesion assay showed that the adhesion of dHL60 cells with Grx1 over-expression to BMSCs was lower than that of the control cells (P<0.01). The results of PCR showed no Grx1 was detected at the whole gene level in Grx1^-/- mice. Compared with the neutrophils from wild-type mice, the neutrophils from Grx1^-/- mice displayed increased adhesion to BMSCs after H₂O₂ treatment. Vascular cell adhesion molecule-1 (VCAM-1) antibody pretreatment induced the adhesion rate back to non-H₂O₂-treated condition. CONCLUSION:ROS promotes the adhesion of neutrophils to BMSCs in bone marrow, which might be regulated by VCAM-1 adhesion signaling-related S-glutathionylation.