Molecular Mechanism Of RhG-CSFF On The Function Of CD4~+T Cell

Objective:Recombinant human granulocyte colony-stimulating factor (rhG-CSF) which has been widely used in allogeneic hematopoietic stem cell transplantation (allo-HSCT) can mobilze stem cells from bone marrow to peripheral blood. Previous studies have demonstrate that rhG-CSF mobilization can affect the number and function of donor T cells greatly. Our previous studies found that the expression of lymphocyte function associated antigen-1 (LFA-1) on CD4+T cells didn’t change during rhG-CSF mobilization. But the adhesive activity of CD4+T cells to intercellular adhesion molecule-1 (ICAM-1) was frustrated after that. Besides adhesion, LFA-1 also plays a central role in the biology of lymphocytes, mediating crucial functional aspects of these cells, including activation, polarization, migration, and signaling. Our previous studies showed that the adhesion polarization and migration of CD4+T have been inhibited after rhG-CSF mobilization.This study aims to investigate the effects of rhG-CSF vitro-stimulation on the adhesion and polarization of CD4+T cells in peripheral blood and the effect of rhG-CSF mobilization on LFA-1/ICAM-1 signaling pathway of CD4+T cells.Methods and Results:(1) The peripheral blood samples were collected from 12 healthy volunteers. Mononuclear cells were isolated from peripheral blood samples by density gradient centrifugation. And CD4+T cells were purified by miniMACS. The purity of CD4+T cells was above 95% detected by flow cytometry.In vitro 200ng/ml rhG-CSF directly acting on the purified CD4+T cells and cultivated for 24h.(2) By using Enzyme standard instrument, we detected the adhesion of CD4+T cells activated by stroma cell-derived factor-la (SDF-la) and ICAM-1. The results showed that the percentage of adhesion CD4+T cells in the experimental group (rhG-CSF acting on the healthy adults volunteers) (61.9± 5.9)% was lower than that from the control group (healthy people) (68.3±7.3)%(P< 0.05).(3) By using inverted microscope,we detected the polarization of CD4+T cells activated by SDF-1α and ICAM-1. The results showed that the percentage of polarizad CD4+T cells in the experimental group (rhG-CSF acting on the healthyadults volunteers) (24.3±4.3)% was lower than that from the control group (healthy people) (47.1±5.1)%(P<0.05).(4) By using western blot,we detected the expression of ZAP-70 and PLCy-1 in LFA-1/ICAM-1 signaing pathway in CD4+T cells after rhG-CSF mobilization. The results showed that the expression of P-ZAP-70 and P-PLCy-1 in CD4+T cells from donors both decreased.Conclusions:rhG-CSF vitro-stimulation may inhibit the adhesion and polarization of healthy people’s CD4+T cells through LFA-1/ICAM-1 signaling pathway. rhG-CSF mobilization may affect the function of CD4+ T cells through the protein kinase ZAP-70 and PLCy-1 that involved in the LFA-1/ICAM-1 signaling pathway.