The Effects Of Trophoblasts On T Cell Proliferation And Cytokine Production And The Underlying Mechanisms

Objectives:To observe the effects of placental trophoblasts on T lymphocyte proliferation and cytokine production and cytokine balance after affected by trophoblast and to explore the possible trophoblast-derived effector molecules.Materials and Methods:Placental trophoblasts were isolated from chorionic villi of normal pregnancy and conditioned medium was made for the culture of T lymphocyte. Peripheral blood mononuclear cells(PBMC)were isolated from healthy female donors and T lymphocytes were purified.Thymidine incorporation test was used to detect the T cell proliferation after the stimulation of phytohemagglutinin(PHA). Radioimmunoassay(RIA)was used to detect estradiol(E2),progesterone(P),human chorionic gonadotropin(hCG)and human chorionic somatomammotropin(hCS)and enzyme-linked sorbent immune assay(ELISA)was used to detect cytokines,TNF-αand IL-10.High performance liquid chromatography(HPLC)was used to measure the concentration of trypotphan in medium.Explant culture of chorionic villi was established to make villi-conditioned medium that was used for proteomic analysis. The protein in the conditioned medium was concentrated,separated with electrophoresis and trypsinized in gel.Shotgun-strategy liquid chromatography-mass spectrometry/mass spectrometry(LC MS/MS)was used to analyze the trypsinized peptide.The raw data of LC MS/MS were searched against human protein database. The identified proteins were grouped according to GOA analysis and searched against the medical literature at Pubmed for their effects on T lymphocyte.Results:(1).hCG,hCS,E2 and P were detected in the conditioned medium,but these hormones were not detected in the un-conditioned medium.The concentration of tryptophan was 19.01±1.04μmol/L in unconditioned medium,3.79±0.45 mol/L in conditioned medium and 15.40±2.66μmol/L in medium when IDO was inhibited by 1-methyltryptophan during previous conditioning.IDO mRNA was expressed in chorionic villi,term placenta and isolated trophoblasts.(2).The proliferation of T lymphocyte was significantly inhibited by the conditioned medium in a dose-dependent manner.The inhibitory effect of conditioned medium on T cell proliferation was partly reversed by either the inhibition of IDO during previous conditioning or supplementation of tryptophan to the conditioned medium.(3).The production of TNF-αwas significantly decreased,while the production of IL-10 was increased,and thus the ratio of TNF-α/IL-10 was decreased by conditioned medium. (4).1869 peptides,corresponding to 548 proteins,were identified in villi-conditioned medium.Among these proteins,as the result of GOA anlysis,285 proteins(52%) were intracellular/membrane proteins,72(13%)extracellular,127(23%)cellular component unknown and 64(12%)miscellaneous.(5).Among the extracellular proteins,31(43%)were plasma proteins,21(29%)extracellular matrix proteins,7 (10%)trophoblast-derived and 13(18)origin-unknown.(6).Ten proteins were found to inhibit T lymphocyte.They were human chorionic gonadotropin(hCG),human chorionic somatomammotropin(hCS),pregnancy-specificβ1-glycoprotein(SP1), α-fetal protein(AFP),glycodelin,transforming growth factor-β2(TGF-β2), thrombospondin-1(TSP-1),pigment epithelium-derived factor(PEDF),macrophage migration inhibitory factor,(MIF)and galectin-1.Conclusions:The product of placental trophoblast inhibited T lymphocyte proliferation,increased the production of IL-10 but decreased TNF-αand subsequently decreased Th1/Th2 ratio.Placental trophoblasts express functional IDO-mediated selective and rapid degradation of tryptophan is one of the mechanisms by which trophoblasts inhibit T cell proliferation.The trophoblast-conditioned medium represented a mixture of several immuneregulatory substances and the inhibitory effect on T cells was the result of synergistic action.