| Backgroud:In pregnancy, hemiallogeneic fetal cells invade the maternal decidua but remain spared from attack by the maternal immune system, posing a great unsolved paradox of immunology. Although several factors have been proposed to explain or contribute to maternal tolerance, the local immunity of decidua play an important role. Studies have shown that the trophoblast cells are surrounded by many immunological cells when they invade into decidua, however, although these immunological cells have potential cytotoxic action, they do not attack trophoblast cells in normal pregnancy. Through the study of characteristic with decidual NK cells, the subset of CD56brightCD16- NK cells are named as decidual natural killer cells (dNK cells). Human uterine endometrium hosts a significant number of NK cells, the percentage of which change during the menstrual cycle and pregnancy. Decidual NK cells begin to increase from secretory phase, which peaks in early pregnancy when dNK cells comprise about 70% of resident lymphocytes and tightly touch with the invading trophoblasts. With the development of human pregnancy, the percentage of dNK cells then fall off, which account about 35% of resident lymphocytes in term decidua. The changes of the percentage and phenotype of dNK cells during pregnancy propose that they may play important roles in implantation and placenta development. Recently the research of origination, phenotype, function and interaction with trophoblasts about dNK cells has been a hot topic in reproduction immunology.Decidual NK cells mainly distribute in decidua which links fetal and maternal interface and which can directly recognize fetal antigen. The specific phenotype and function of dNK cells may contribute to keep immunotolerance in fetal-maternal interface, also can they secrete an array of cytokines facilitate decidual blood rebuilding and maintain the balance of Th1/ Th2 cytokines. Study the function and status of dNK cells undoubtedly help us to further comprehend the physiological phenomena of pregnancy and also can provide new ways to research the mechanism of pathological pregnancy, as well as the development in reproduction immunology.Objective:To detect the distribution of dNK cells and compare the percentage, phenotype, Th1/ Th2 cytokine secretion and cytotocxic action between dNK cells and pNK cells, which can help us to further approach the important interaction between immunotolerance in fetal-maternal interface and dNK cells.Methods:1) Third-trimester decidua and peripheral blood was obtained during elective caesarean sections (n = 20), the method of IHC was used to detect the distribution of dNK cells.2) Cell suspensions were prepared by an electromechanical dispersal method and centrifugated using a standard gradient sedimentation technique. The percentage and phenotype of dNK cells and pNK cells were detected by flow cytometric analysis.3) The method of RT-PCR was used to detect the mRNA expression of HLA-G, HLA-E and MICA , which are ligands of NKG2A and NKG2D respectively.4) Flow cytometry was used to detect the expression of intracellular Th1/Th2 cytokines IFN-γand IL-4 in dNK cells and pNK cells.5) dNK cells and pNK cells were separated by MACS from PBMC and DBMC, whose purity was then detected by Flow cytometry. The cytotoxic activity of dNK cells and pNK cells against targets was measured in a standard 4-hr 51Cr-release assay.Results:1) The result of IHC showed that the distribution of dNK cells was distinct, many of which distributed at fetal-maternal interface.2) Comparede with pNK cells, dNK cells are the dominant group of decidual lymphocytes.3) The expression of CD16 were significantly decreased on dNK cells as compared with those on pNK cells, as we all know, expression of CD16 is one of important signals to measure cytotoxic activity; however, it was unclear that CD69 expression were significantly increased on dNK cells as compared with those on pNK cells, which seemed paradoxical to the theory of immunotolerance in fetal-maternal interface, because that CD69 is the marker of earlier period activation.4) Decidual natural killer cells had high expression of NKG2A, which ligand HLA-G and HLA-E mRNA were also expressed in trophoblast tissue. Although dNK cells highly expressed NKG2D, there was no MICA mRNA expression.5) Compared with pNK cells, the expression of intracellular Th1/Th2 cytokines IFN-γand IL-4 were significantly high in dNK cells. IFN-γand IL-4 are the most important cytokines in regulating Th1/Th2 immunological balance, suitable quantity of which play an important role in regulating immunological balance at fetal-maternal interface.6) Although there is much perforin and granzyme in dNK cells, which propose they have potential cytotoxic action, their cytotoxic activity of dNK cells against targets were significantly decreased than pNK cells, maybe the cytotoxic activity of dNK cells were suppressed by something at fetal-maternal interface . Conclusions:Decidual natural killer cells are the important natural lymphocytes, whose percentage, phenotype, Th1/Th2 cytokine secretion and cytotocxic action are obviously different from peripheral NK cells, also they tightly touch with the invading trophoblasts. dNK cells and their secreting cytokines compose the center of decidual immunological regulation through development, regulation, receptor expression, biological effect. From the above results, we conclude that dNK cells play an important role in regulating immunological tolerance at fetal-maternal interface.
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