Effects Of Neuropeptide Substance P On Migration And Expression Of Chemokine Receptors Of NK-92MI Cells

Objective: Natural killer(NK)cells are indispensable defense cells of the immune system.NK cells can kill invasive pathogens by cytotoxicity and play a key role in tumor immunity.NK cells can accumulate in many organs and peripheral blood,such as spleen,lung,liver and uterus.When the body is infected or cancerous,NK cells in tissues and peripheral blood will be recruited to inflammatory areas and cancerous tissues by chemotaxis of various chemokines which can regulate the migration of NK cells.SP,a neuropeptide secreted by the nervous system,mainly involved in the neuroregulation,as well as immunoregulation and inflammatory responses.SP is closely related to the immune system,which can promote the proliferation of NK cells and activate many kinds of immune cells.SP is an essential regulatory polypeptide in human body and plays an important role in the recruitment of immune cells during inflammations.NK cells can be regulated by various chemotactic substances,which initiated the migration of NK cells to kill pathogens and malignant cells by cytotoxicity.In this study,we investigated the effects of SP on the migration of NK92-MI cells and the expression of chemokine receptor on NK92-MI cells,in order to explore the direct or indirect regulation on the migration activity of NK cells by SP.The effects of SP on the expression levels of CCR7,CXCR3 and CXCR4 in NK92-MI cells were analysis,as well as the role of NK-1R in regulating the migration activity of NK92-MI cells by SP,to further discuss the mechanism of regulating the migration activity of NK cells by SP.Methods:(1)Transwell test was used to detect the effects of SP in different concentrations(10-12 mol/L,10-10 mol/L,10-8 mol/L and 10-6 mol/L)on the migration capability of NK92-MI cells,and to detect the impacts in chemotaxis of chemokine CCL21 and CXCL12 on NK92-MI cells by SP.(2)Real-time PCR was used to determine the m RNA levels of chemokine receptors CCR7,CXCR3 and CXCR4 in NK92-MI cells treated by SP in different concentrations.(3)Surface expression of CCR7,CXCR3 and CXCR4 on SP-treated NK92-MI cells was measured by Flow cytometry.(4)NK92-MI cells were treated with NK-1R antagonist,then the migration was detected using Transwell test,and m RNA expression of CCR7,CXCR3 and CXCR4 was determined by Real-time PCR.Results:(1)SP has direct chemotaxis for NK92-MI cells.SP in the concentrations of 10-12 mol/L~10-10 mol/L gradually enhance the migration of NK92-MI cells according the increase of SP concentrations.SP at a concentration of 10-10 mol/L promotes the maximum migration of NK92-MI cells.Then the SP concentration continued to increase,but the promoting migration gradually weakened.SP at different concentrations can also enhance the chemotactic effect of chemokines CXCL12 and CCL21 on NK92-MI cells.Treatment of SP at lower concentrations(10-12 mol/L~10-10 mol/L)gradually enhance the chemotaxis of CXCL12 and CCL21 on NK92-MI cells,while the enhanced chemotaxis of CXCL12 and CCL21 on NK92-MI cells was reduced when treated by SP at higher concentrations(10-8 mol/L~10-6 mol/L).(2)SP can significantly increase the m RNA expression of CCR7,CXCR3 and CXCR4.Treated by SP in the concentrations of 10-12 mol/L ~ 10-10 mol/L for 6h,m RNA expression of CCR7、CXCR3 and CXCR4 are all augment.Only in group of 10-12 mol/L,m RNA level of CXCR4 slightly increased,which was not statistical significance compared with the control group.Treatment of SP in the concentrations of 10-12 mol / L~10-6 mol / L for 12 h can persistently promote the m RNA expression of CXCR3 and CXCR4,but there was no significant increase in the m RNA expression of CCR7.(3)Effects of SP in concentrations of 10-12 mol/L~10-6 mol/L on the surface expression of CCR7,CXCR3 and CXCR4 are distinct.The levels of CCR7 were increasing with the raise of SP concentrations,and there are significantly increased CCR7 levels in groups of 10-8 mol/L and 10-6 mol/L.After treatment of SP in different concentrations,the augment of CXCR3 was not significant compared with the control.Following the increase of SP concentrations,expression of CXCR4 firstly increased,and then decreased.SP at concentrations of 10-10 mol/L and 10-8 mol/L significantly promoted the expression of CXCR4.The trend of SP increasing the expression of three chemokine receptors was not consistent with the effects of SP on migration ability of NK92-MI cells,suggesting that there are competitive expressions of various chemokine receptors,and/or there also be other chemokine receptors synergies.(4)After treatment of NK-1R antagonist,SP,CCL21 and CXCL12 could not enhance the migration of NK92-MI cells,and SP could not promote the m RNA expression of CCR7,CXCR3 and CXCR4,which implied that NK-1R antagonists play a blocking role.Conclusion: SP can directly promote the migration of NK92-MI cells,indicating that SP has direct chemotactic effect on NK cells.SP can increase the expression of chemokine receptors CCR7,CXCR3 and CXCR4 on NK92-MI cell,and enhance the chemotactic effect of CXCL12 and CCL21 which are the ligands of these chemokine receptors on NK92-MI cells.This indicated that SP could indirectly regulate the migration activity of NK cells by up-regulating the expression of certain chemokine receptors.The regulation of SP in migration ability of NK92-MI cell is dependent on NK-1R.