Preliminary Study On NKT Cells Activation And The Mechanisms Of Abortion In C57BL/6J Early Pregnant Mice

Backgroud Natural killer T cells(NKT) are an evolutionary conserved sub-lineage of T cells that bridge the innate and adaptive systems and characterized by reactivity to self-glycolipids andα-galactosylceramide(α-Galcer) that are presented by monomorphic MHC class-Ⅰ-like molecule CDld.Upon antigen recognition,NKT cells can rapidly produce multiple cytokines and up-regulate cytotoxic molecules,α-Galcer can activate NKT cells and induce abortion in early pregnant mice when injected i.p..Ito and others found that NKT-knock-out mice can not be mediated abortion byα-Galcer.And this discovery was published in PNAS(2000).Kinjo and others examined compounds from Sphingomonas bacteria,Gram-negative bacteria which are highly abundant in the environment and found most mouse NKT cells recognize glycosphingolipids from Sphingomonas,whose structure isα-Galcer analogues, through CD1d binding.And this discovery was published in nature(2005).The recent discovery of pregnancy hormone,progesterone in particular,can regulate antibody production,decrease oxygen burst in monocytes and reduce proinflammation cytokines caused by bacterial products.That may be the role of immune regulation.Objective we studied to optimize a method for expanding mouse NKT cells in vitro and searched the effect of specific antigen or varies of cytokines on the expansion.Then we determined the properties of expanded NKT cells.Meanwhile, we evaluated changes of immune function and mechanisms ofα-Galcer induced early abortion in a mouse model.Furthermore,the role of MPA in preventing abortion started byα-Galcer was preliminary explored in this study.Methods Mouse splenic singal nuclear cells were isolated and cultured.Drug sensitivity toα-Galcer was analyzed with MTT and PI stain.Then the effect ofα-Galcer and rhIL-2 on cell expansion capability was performed.The activated or not NKT cells were selected by flow cytometrx(FACS),then the changes of ultrastructure of NKT cells were observed with transmission electron microscopy.Cytotoxic activation of NKT cells was detected by MTT assay. Pregnant C57BL/6J mice were injected i.p.withα-Galcer to activate NKT cells and rate of resorption was examined.The expressions of bcl-2 and PR in the decidua was examined by immunohistochemical staining with specific monoclonal antibodies.when MPA was injected i.h.2h beforeα-Galcer administration,the rate of resorption was examined and effect of MPA was explored.Results At 24h after treated with 50～100 ng/mLα-Galcer,the proliferation activity of test NKT cells were significant higher than that of control(P＜0.05). And the best concentration was 100ng/mL.The rate of hypodiploid splenocytes increased when drug consentration rose.Once the consentration exceeded 200 ng/mL,the rate of that was significantly higher than control.Each of 3 cases of MNCs was divided into 4 groups.During 12d culture,cells from Groupα-Galcer and control were rapidly incurred apoptosis,a-Galcer+cytokine did better than cytokine only(P＜0.05).At the 4th,8th,12th day,the total number of spleen NKT cells was 1.6,2.8 and 3.4 times higher than control.Activated NKT cells displayed typical morphological characteristics of activated cells,and apoptosis NKT cells showed morphological changes including chromatin condensation,apoptotic body, and broken nuclear membrane.In 8-hrs MTT cytotoxicity assays,the anti-tumor effect of activated NKT cells was found in cell line HL60 but not in NK cell-sensitive cell,K562.Afterα-Galcer administration,the rate of abortion was significantly higher and the expression of bcl-2 in decidua was extremely lower than that of control.MPA could reduce this effect.Conclusions In this study we had established culture conditions containingα-Galcer that allow for the short-term proliferation of large numbers of NKT cells from mouse spleenic MNCs.IL-2 was the essential cytokines for the expansion of NKT cells.Uponα-Galcer stimulation,NKT cells performed effect of secreting cytokines and cytotoxicity. The effect ofα-Gal cer induced abortion in C57BL/J mice might associate with direct function of NKT cells and their potency of secreting cytokines.In addition to the important role of NKT cells,the pharmacological effects ofα-Galcer seemed not to be ruled out.The ability of MPA to modulate the immune response might be a critical mechanism by which progestins prevent pregnancy loss.