Autocrine And Paracrine Regulation Of Chemokine CCL2 In The DSC Functions And Th2 Bias At Human Materno-fetal Interface

Research Background Chemokines, chemotactic cytokines, are an array of small molecules of heparin-binding proteins that direct the trafficking of circulating leukocytes into the sites of inflammation or injury. The chemokines and their receptors are now involved in pathogenesis of a variety of diseases. To date, the materno-fetal interface has been found to produce a lot of chemokines, and express the corresponding receptors. Our previous studies have demonstrated that human first-trimester trophoblast cells can produce SDF-1 and CXCL16, and express their receptors CXCR4 and CXCR6. Moreover, decidual CD56^bright CD16^- NK cells expressed CXCR4, andγδT cells expressed CXCR6 highly. The first-trimester human trophoblast cells has been able to induce proliferation and invasion of themselves in an autocrine manner, and recruit the CD56^bright CD16^- NK cells andγδT cells homing into decidua by expressing SDF-1/CXCR4 and CXCR6/CXCL16, respectively.During pregnancy, the decidual tissue, the maternal component at the interface, is composed predominantly of decidual stromal cells (DSC). Not only is the DSC classically considered to be nutritious to embryo because of their high glycogen content, but also involved in a series of immune regulations such as production of cytokines, and antigen phagocytosis and presentation. A large and specific population of immune cells infiltrate constitutively into the decidua. Therefore, the decidua is in closest contact with the fetal-derived trophoblasts and decidual lymphocytes, and thought to be a place where immune regulations between mother and fetus take place. So we proposed that the decidua may be involved in the materno-fetal tolerance by secreting chemokines and expressing their corresponding receptors. MCP-1, monocyte chemotaxis protein-1, CCL2, is a CC chemokine that attracts monocytes, memory T lymphocytes and natural killer cells in vitro by way of binding its receptor CCR2. Lately, CCL2 has been demonstrated to be involved in Th2 polarization, and a successful pregnancy is a Th2 phenomenon. Here we found that human first trimester decidua and decidual stromal cells expressed high levels of CCR2 and CCL2. Therefore, CCL2 may play a role in Th2 phenomenon at materno-fetal interface.Objectives Based on the research above, biological functions of human decidual stromal cells were explored in secretion of CCL2 and expression of CCR2, which might be involved in autocrine regulation on the DSC functions, and paracrine regulation on the Th2 bias at human materno-fetal interface.1. Transcriptional characteristics of chemokine receptors and the correspondent ligands in decidua and decidual stromal cell of human first trimester gestationMethods The deciduas were from women who had undergone an artificial abortion at 5-11 weeks of normal gestation for non medical cause. The total RNA was extracted by using the TRIzol reagent, from decidua or the Percoll-gradient-isolated DSC. The expression of chemokine receptors in the decidua and DSC were investigated by way of semi-quantitative RT-PCR.Results The chemokine receptors, CCR2, CCR5, CCR10, were highly expressed in human decidua and DSC, while CCR3, CCR6, CCR8-9, CXCR1, and CXCR4 were moderately expressed in human decidua. The chemokine receptors CCR1, CCR4, CCR7, CCR9, CXCR3, and CXCR5-6 were expressed only in the partly decidua, while no CXCR2, XCR1, and CX₃CR1mRNA were found in any decidua tissue. The freshly isolated DSC expressed moderately CCR7, CCR9, CCR1, CCR3, CCR4, CCR6, CXCR6, XCR, and CX3CR1. The CCL2 and CCL13, ligands of CCR2, and CCL28, that of CCR10, were expressed highly in the decidua and DSC.Conclusion The chemokine receptors CCR2 and CCR10 that were expressed by decidua and DSC may play an important role in the cell constitution at materno-fetal interface in human first trimester pregnancy.2. Expression of chemokine CCL2 and its receptor CCR2 in human decidua and decidual stromal cells in the first trimester pregnancyMethods The expressions of CCR2 and its ligand CCL2 in decidua and DSC were determined by way of semi-quantitative RT-PCR, immunostaining, flow cytometry, respectively. The secretion of CCL2 was detected by ELISA.Results The results showed that CCR2 and its ligand CCL2 were highly expressed in decidua and DSC. The DSC could secret CCL2 in a time-dependent manner, and the highest concentration was 21.72±2.34ng/ml. About 34% CD56⁺CD16^-CD3^- decidua NK, 50%CD4⁺ T cells and 66% CD14⁺ monocytes expressed CCR2.Conclusion The CCR2 and CCL2 were expressed in decidua and DSC of human first trimester gestation, which might play an important role in the decidual immune cell constitution, and the cross-talking between decidua stromal cells and decidua immunocompetent cells in human early pregnancy.3. The regulation of CCL2/MCP-1 secretion and autocrine regulation on human decidual stromal cell of the first-trimester gestationMethods The viability of DSC was analyzed with MTT. The primary DSCs were treated in vitro by the pregnancy-associated hormones, E2, progesterone and hCG, or Th1 and Th2 cytokines. The CCL2 expression and secretion was determined respectively by real time PCR and ELISA.Results All the pregnancy-associated hormones, E2, progesterone and hCG, did promote CCL2 transcription and secretion by DSC. U0126, an inhibitor of MAPK, and RS 102895, antagonist of CCR2, inhibited CCL2 secretion by DSC. Th2 cytokine, IL-4, and Th1 cytokine, IFN-γ, also increased CCL2 secretion. IL-4 and TNF-αhad synergistic effect on CCL2 production by DSC, but IL-4 and IFN-γdid not. The rhCCL2 could enhance the proliferation and viability of human DSC of the first-trimester gestation.Conclusion CCL2 may stimulate human DSC viability in autocrine manners. And Th1/Th2 cytokines, pregnancy-associated hormones could regulate CCL2 secretion of DSC4. Decidual stromal cells maintained Th2 predominance at materno-fetal interface through producing and secreting CCL2Methods DIC were treated by different stimuli, including rhCCL2, rhCCL2+CCL2 antibodies, the DSC-derived CM, CM+CCL2 antibodies, CCL2+U0126 and CpG used as positive control. Then the apoptosis and proliferation of DIC was detected respectively by Annexin V/FITC and PCNA with FCM, and the secretion and intracellular expression of Th1 cytokines, TNF-αand IFN-γ, and Th2 cytokines, IL-4 and IL-10, were determined by CBA and FCM, respectively. Transcription of GATA-3 and T-bet in DIC was evaluated by Real time PCR.Results The results showed that rhCCL2 and the DSC-derived CM could stimulate DIC proliferation and inhibit DIC apoptosis. The rhCCL2 and the DSC-derived CM promote Th2 cytokine, IL-4 and IL-10 production, and inhibit secretion of Th1 cytokine, TNF-αand IFN-γ. The CCL2 and CM could also increase GATA-3 transcription and suppress T-bet transcription in DIC.Conclusion Decidual stromal cells can secret CCL2 that in turn increases GATA-3 transcriptions of DIC, which leads to Th2 polarization at materno-fetal interface.