| Chemokines are a large family of small soluble cytokines, and are well known for their crucial roles in immune system. So far, about50chemokines and18different chemokine receptors have been identified in humans. According to the relative position of cysteine residue, chemokines can be divided into four main groups:CXCL, CCL, XCL, and CX3CL subfamilies. Eotaxins belong to CCL chemokine family. They are broadly recognized as eosinophils’high selectively chemoattractants. CCL24is the second member of eotaxins, and is also called eotaxin2or MPIF-2(myeloid progenitor inhibitory factor2). Via binding with CCR3, a seven transmembrane spanning receptor, CCL24mainly mediates eosinophil trafficking, and participates in atopic disorders, parasitic infections, systemic diseases etc. It has been reported that CCR3is highly expressed at the maternal-fetal interface; eotaxin1, the nearest relative of CCL24, is also detected there. Therefore, we propose that chemokine CCL24is expressed at the maternal-fetal interface, and the CCL24/CCR3axis may participate in embryo implantation, decidualization, placentation, and immune tolerance at maternal-fetal interface. In this study, we analyzed the expression of CCL24and CCR3at maternal-fetal interface in human first-trimester pregnancy, and investigated their functional regulation in the crosstalking interface.Part Ⅰ. CCL24regulates the biological function of trophoblasts in an autocrine mannerObjective:To test expression level of CCL24and CCR3in the surface of trophoblasts, and then investigate the potential role of CCL24/CCR3axis at the maternal-fetal interface.Methods:Isolation, purification and characterization were for human first-trimester trophoblasts. IHC (Immunohistochemistry), ELISA (Enzyme-linked immunosorbent assay) and FCM (Flow cytometry) were used to detect the expression of CCL24and CCR3in trophoblasts. The expression of CCL24and CCR3in trophoblasts were investigated in co-culture with DSCs (Decidual stromal cells). Trophoblasts were in vitro treated with rhCCL24(recombined human CCL24) for48hours, and then BrdU proliferation assay was utilized to analyze their proliferation. MTT colorimetric assay and Transwell invasion assay were used to evaluate their viability and invasion ability.Results:By IHC, the cytomembrane and cytoplasm of trophoblasts were moderately stained for CCL24and highly stained for CCR3in all villi samples. The secretion level of CCL24showed a peak at24h, and then decreased with duration along. CCR3protein could be detected on the surface of trophoblasts10.79±0.82%. In DSCs and trophoblasts co-culture, the CK7positive trophoblasts accounted for about20%of final cells. The secretion of CCL24by trophoblasts increased from800pg/ml to1400pg/ml (P<0.01). Meanwhile, the expression of CCR3on trophoblasts (57.15±11.27)%was also significantly elevated (P<0.01) in the co-culture for48h. CCL24promoted the proliferation and viability of trophoblasts (P<0.01). In addition, CCL24inhibited the invasion ability of trophoblasts in dose-dependent manner; higher concentration had more effect.Conclusion:Trophoblasts secrete CCL24, and also express receptor CCR3. Trophoblasts express higher level of CCR3and may also secrete more CCL24in coculture with DSCs. CCL24promotes the proliferation and viability but suppresses the invasion of trophoblasts in dose dependent manner, which means a complicated regulation for embryo implantation.Part Ⅱ. Trophoblast-derived CCL24regulates the biological function of DSCs in a paracrine mannerObjective:To test the expression of CCL24and CCR3on the surface of DSCs, and then investigate the potential effect of CCL24/CCR3axis on the biological function of DSCs.Methods:IHC, ELISA and FCM were used to detect the expression of CCL24and CCR3in DSCs. The expression of CCR3in DSCs was investigated in co-culture with trophoblasts. We treated DSCs with pregnancy-associated hormones (estrogen, progesterone or HCG), and then used FCM to investigate the expression of CCR3on the surface of DSCs. We treated DSCs with rhCCL24, then BrdU proliferation assay was performed to analyze their proliferation, Annexin V/PI assay was used to examine their apoptosis, cell counting was used to estimate the general effect of CCL24on the number of DSCs.Results:By IHC, CCL24staining was weak or not observed in decidual epithelial and stromal cells, while CCR3was highly stained in all decidual epithelial and stromal cells. From24h to96h, we did not observe the secretion of CCL24by DSCs at any time point. By FCM,(12.89±6.3)%of DSCs were CCR3positive. When co-cultured with trophoblasts, the expression of CCR3on DSCs increased significantly, and reached to (33.87±0.60)%. HCG promoted the expression of CCR3on DSCs, especially at the lower concentration (P<0.01); both17β-estradiol and progesterone increased the expression of CCR3on DSCs. CCL24promoted the proliferation of DSCs as well as their apoptosis. CCL24up-regulated the number of DSCs, and exhibited the maximum effect at the concentration of10ng/ml (P<0.01). Meanwhile, the change could be counteracted by neutralizing antibody a-CCL24and a-CCR3.Conclusion:DSCs express CCR3, but don’t secrete CCL24. The trophoblast-derived CCL24may regulate the biological function of DSCs in a paracrine manner. Trophoblasts and DSCs interaction stimulates the expression of CCR3on DSCs, and may promote the action of CCL24in decidualization. Pregnancy-associated hormones up-regulate the expression of CCR3on the surface of DSCs. In human first-trimester pregnancy, the increased steroid hormones may also promote the effect of CCL24on functions of DSCs. CCL24promotes the proliferation as well as the apoptosis of DSCs.Part Ⅲ. CCL24regulates the function of γδT cells at the maternal-fetal interface in human first-trimester pregnancyObjective:To test the composition of DICs and their expression of CCR3. To detect the expression of co-stimulatory molecules, the proliferation or apoptosis associated molecules in γδ T cells. To evaluate the potential effect of CCL24/CCR3axis on the biological function of y8T cells.Methods:DICs were isolated from decidua, then their composition and expression of CCR3were tested by FCM. We purified y8T cells from DICs, detected the expression of nine co-stimulatory molecules on the surface of the cells, and then evaluated the regulatory function of CCL24on the co-stimulatory molecules onyS T cells. We tested the expression of proliferation and apoptosis-associated molecules, and evaluated the regulatory role of CCL24on the those molecules.Results:The main components of DICs were CD3" CD56+NK cells, which accounted for60%; both CD14+Mcp cells (macrophage) and CD3+T cells were about10-20%; the rest were a small subset of DC cells and other unmarked cells. About half of T cells in DICs were CD3+γδ T cells. As measured by FCM, CCR3protein could be detected in all the DICs. T cells, including γδ T cells, from both DICs and PBMC expressed high level of CCR3.By magnetic isolation, the purity of decidual γδ T cells was about90%. About (8.62+4.78)%of y8T cells were CD69+. Among nine co-stimulatory molecules, ICOS, GITR, CD40L and NKG2D were highly expressed; OX-40and PD-1were moderately expressed; while the level of CTLA-4, CD28, and NKG2A were very low. Both rhCCL24or the culture supernatant of trophoblasts inhibited the expression of ICOS, GITR, and PD-1. When a-CCL24and a-CCR3were added to the supernatant, the inhibitory function of CCL24was suppressed.We also detected level of Ki67, Bcl2, and Fas, whereas FasL is at low level. After treated γδ T cells with rhCCL24or cell culture supernatant of trophoblasts, the expression of Ki67and Bcl2in y8T cells was inhibited. Meanwhile, when a-CCL24and a-CCR3were added to the culture supernatant, the inhibition could be suppressed.Conclusion:CCR3is highly expressed on γδ T cells, and almost all DICs express CCR3. The combination of CCL24and CCR3may regulate the biological function of DICs in a paracrine manner. The surface of γδT cells is rich in ICOS, GITR, CD40L and NKG2D, and medium level of OX40and PD-1, and lacks CD28, CTLA-4and NKG2A. CCL24may hamper the expression of GITR, ICOS and PD-1, and regulate the activation of γδ T cells. The y8T cells express medium level of Ki67, Bcl2and Fas. CCL24inhibits proliferation-associated molecules Ki67and Bcl2. The CCL24/CCR3axis may regulate γδ T cells functions during first-trimester pregnancy. |
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