The Role Of Apolipoprotein M-Sphingosine-1-Phosphate Axis In Inflammation Of Macrophage

Objective : To define the expression of apo M in peripheral blood mononuclear cells(PBMCs). Investigate the role of apo M-S1 P axis in inflammation of macrophage and its perhaps molecular mechanisms involved.Methods:PBMCs were isolated from healthy adult volunteer’s whole blood by ficollhypaque density gradient centrifugation. Then the cells were labeled with different antibodies such as CD3, CD19, CD14, CD56 and CD16 which were the surface markers of T-cells, B-cells, NK-cells and monocytes, respectively. Apo M was labled by apo M monoclone antibody with PE fluorescent dyes. The expression of apo M in PBMCs was further observed under laser scanning confocal microscope. The expression ratio of apo M in different cell subsets was detected by Flow Cytometry method.Taking THP-1 cells as the research object, eatablish a in vitro cell model of inflammation by inducing THP-1 cells into macrophages after incubating for 24 hours with 100ng/ml PMA. Grouping method:(1)control group;(2)the macrophages were solely pretreated with apo M rencombination protein(rhapo M,10μg/ml) for 3 hour;(3)the macrophages were pretreated with rhapo M(10μg/ml) for 3 hour, then were incubated with culture medium containing LPS 10ng/ml for 6 hours.(4)the macrophages were solely treated with LPS(10ng/ml) for 6 hours.Macrophages were pretreated with S1P2 antagonist JTE-013(30μM), agonist CYM-5520(1μM、10μM、50μM), S1P1、S1P3、S1P4、S1P5 agonist FTY720(10μM), respectively. Then cells were incubated with culture medium(containing rhapo M 10μg/ml) for 9 hours. The levels of m RNA of TNF-α and IL-1β were detected by the dual fluorescent quantitative real-time polymerase chain reaction.Result:Confocal microscopy results showed that apo M was expressed on T cells, B cells, NK cells and monocytes, respectively. Flow cyto metric analysis showed that the expression ratio of apo M was 14.4% on CD14+ monocytes. The expression ratios of apo M on CD3+T cells, CD19+B cells, CD56+ NK cells and CD16+NK cells were 7.4%, 6.2%, 2.4% and 4.2%, respectively. Compared with the LPS treatment alone group, the m RNA levels of TNF-α and IL-1β were significantly up-regulated by 15% and 92% after pretreating with apo M(P=0.0383, P=0.0049). In vitro, rhapo M could promote the expression of TNF-α and IL-1β(109% and 35%,P=0.0007 and P=0.0026). Compared with the rhapo M treatment alone group, the m RNA levels of TNF-α and IL-1β were significantly up-regulated by 86% and 65% after pretreating with JTE-013(P<0.0001). Otherwise the m RNA levels of TNF-α and IL-1β were significantly down-regulated by 41% and 24% after pretreating with CYM-5520(P=0.0406 and P=0.0275). Double factor analysis of variance showed that there was interaction between apo M and JTE-013(P<0.01). In vitro, the m RNA levels of TNF-α and IL-1β were dramatically inhibited by FTY720(48% and 60%,P<0.0001). Double factor analysis of variance showed that there was no significant interaction between apo M and FTY720( P>0.05).Conclusion:Apo M was expressed on T cells,B cells,NK cells and monocytes.Apo M rencombination protein could up-regulatethe m RNA levels of TNF-αand IL-1βin LPSinduced inflammation.Apo M rencombination protein might promote the inflammation via S1P2 signaling pathway.