The Change Of S1P/S1P Receptor Signal Of CD4~+T Cell After G-CSF Mobilization And The Role Of S1P5 On LFA-1 Adhesion

Background and aims:Granulocyte Colony-Stimulating Factor (G-CSF) plays a role in the regulation of immunity. We found previously that G-CSF mobilization decreased the adhesion of CD4+ T cells to ICAM-1 and increased the SphK activity of monocytes, though G-CSF mobilization did not impact SIP concentration in donors' plasma. It is reported that Lymphocyte function antigen-1 (LFA-1) is essential for biological functions of lymphocyte, Sphingosine 1-phosphate (SIP) regulate LFA-1 mediated adhesion and SIP antagonist mediated lymphocyte homing depends on LFA-1. Therefore, we speculated S1P/S1P receptor signal may be involved in G-CSF immunoregulation network. Thus, we investigated the changes of S1P/S1P recepter signal after G-CSF mobilization, the role of S1P5 in LFA-1 mediated adhesion and the expression of G-CSF receptor on CD4+T cells in this article aiming to explore the role of SIP/SIP receptor signal in G-CSF-mediated immune regulation.Methods:(1) On CD4+T cells before and after G-CSF mobilization, the expression of S1P5 was detected by Realtime RT-PCR and Flow cytometry separately, migration to SIP was detected by Transwell, the affect of SIP on LFA-1 was detected by adhesion assay, and the phosphorylation of LFA-1 was detected by western blot. (2) We used lovastatin to block LFA-1 and SiRNA targeted S1P5 to silence gene expression on Jurkat. Proliferation was detected by CCK-8, apoptosis was detected by PI and Annexin V stain and the expression of S1P5 was detected by Realtime RT-PCR. (3) ConA was used to stimulate CD4+T cells. The expression of G-CSF receptor was detected by Flow cytometry. Affect of G-CSF on proliferation of CD4+T cells and Jurkat cells was detected by CCK-8.Results:(1) After G-CSF mobilization, the expression of S1P5 on CD4+T cells was increased, and other S1P receptors were not affected by G-CSF mobilization. On protein level, the expression of S1P5 was upregulated in CD4+T cells, CD8+T cells and NK cells. CD4+ T cells migration toward SIP was reduced after G-CSF mobilization. S1P did not affect the LFA-1-mediated adhesion of CD4+T cells to ICAM-1 before G-CSF mobilization, but SIP reduced the LFA-1-mediated adhesion of CD4+T cells to ICAM-1 after G-CSF mobilization. Phosphorylation of LFA-1 on CD4+T cells was reduced after G-CSF mobilization. (2) The proliferation and the gene expression of S1P5 of Jurkat were inhibited by LFA-1 inhibitor. Silencing S1P5 gene did not affect proliferation and apoptosis of Jurkat cells and phosphorylation of LFA-1, but adhesion of CD4+T cells was enhanced. (3) G-CSF receptor mRNA was detected before and after G-CSF mobilization. The expression of G-CSF receptor induced by ConA was reduced by G-CSF mobilization in vivo. But there was no the same affect in vitro. G-CSF could inhibit proliferation induced by ConA in vivo and in vitro. G-CSF inhibited proliferation of Jurkat cells.Conclusions:The expression of S1P5 on CD4+T cells was increased after G-CSF mobilization, which might lead to the reduction of CD4+T cells migration toward S1P and negatively regulate the adhesion mediated by LFA-1. G-CSF might modulate CD4+T cells function directly through G-CSF receptor.