| Chemokines are a super family of cytokines of 8 to 14 kDa that play important physiological roles by binding to specific G-protein-coupled receptors. Fifties chemokines and 18 chemokine receptors have been identified as to now. Chemokine CXCL16 was discovered recently as a ligand for the CXC-chemokine receptor CXCR6, and described as a scavenger receptor for oxidized low density lipoprotein when expressed on macrophage membrane. We have demonstrated that CXCR6 is transcribed highly in human trophoblasts of first-trimester, but to date the regulatory roles of the chemokine pair CXCL16/CXCR6 at materno-fetal interface remains elucidated. In this study, we are to first analyze the expression of CXCL16/CXCR6 in the main functional cells at human materno-fetal interface, and then investigate their biological functions at materno-fetal interface.1. Isolation, purification and characterization of human first-trimester trophoblast and decidual functional cellsAs a key cell of placenta, isolation, purification and characterization of villous cytotrophoblasts (VCT) and extravillous cytotrophoblasts (EVCT) were established previously by our group.Decidual stromal cell (DSC) population accounts for 70% in all decidual cells, and decidual immune cells (DIC) accounts for 15%. We described here a simple procedure to isolate decidual stromal cells and decidual immuocytes at one time from deciduas by enzyme digestion, Percoll gradient centrifugation, and short period culture. The homogeneity of the stromal cells was almost 100% as verified by immunocytochemistry of the combined vimentin, keratin, muscle actin intermediate filaments and coagulation factor VII. The cell types of decidual immunocytes were analyzed by three-color, two-color and one-color fluorescence-marked flow cytometry. The results showed that decidual CD56~+CD16~-NK cells accounted for 67.02±18.33%,monocytes accounted for 5.28±0.29% and NKT cell accounted for 2.35 ± 1.62%, respectively, in all the decidual immunocytes. T cell population in peripheral blood was decreased significantly in early pregnant than that of non-pregnant control at luteal phase (p<0.05), but the population of NK cells, NKT cells, and monocytes in the peripheral was similar to that of the control. In a whole, we established a procedure to separate and identify DSC and DIC from normal human deciduas successfully, which provided basis for our research as follow.2. The expression of CXCL16 and CXCR6 at materno-fetal interface of human first trimester pregnancyThe transcription and translation of chemokine CXCL16 and its receptor CXCR6 in the first-trimester trophoblasts and decidual stromal cells (DSC) were first analyzed by RT-PCR and immunostaining, respectively. It was found that both CXCL16 and CXCR6 were highly transcribed and translated in the villous cytotrophoblasts (VCT), extravillous cytotrophoblasts (EVCT) and syncytiotrophoblast (ST). However, the mRNA level of CXCL16 and protein levels of CXCL16 and CXCR6 were very low although the transcription of CXCR6 was high in DSC, which suggested that CXCL16/CXCR6 was impossible to regulate the function of DSC at materno-fetal interface. Thereafter, we analyzed the expression of CXCR6 on the decidual immunocytes (DIC) using three-color flow cytometry. The positive rate of CXCR6 was 87.29±6.95% on γδΤ cells, 47.71±6.04% on monocytes, 44.14±4.40% on NKT cells, 30.39±5.23% on T lymphocytes. The deciudal CD56~+CD16~-NK cells and CD56~+CD16~+NK cells expressed CXCR6 in very low level, 4.79±1.30% and 4.84±0.77% respectively. Hence, CXCR6 and CXCL16 were expressed selectively in the functional cells at the materno-fetal interface.3. CXCL16 was secreted by trophoblasts and induced the proliferation and invasion of trophoblast in an autocrine manner.The interaction of CXCL16 to CXCR6 may stimulate CXCR6-positive cell proliferation in an autocrine or paracrine manners. Thereafter, we carried out a series of experiments to demonstrate possible regulation of CXCL16 in trophoblast biological functions. The result detected by ELISA showed that trophoblasts were verified to secret CXCL16 spontaneously and continuously in vitro, when they were seeded in 1 ×10~6 cells/ml, the accumulated concentration of CXCL16 was 2.690±0.180 ng/ml in culture for 100 h. After treatment with INF-γ, CXCL16 synthesis and secretion by trophoblasts were increased significantly (p<0.01). But the ADAM10treatment only increased the shedding of CXCL16 from trophoblast.After human trophoblasts had been treated with rhCXCL16, the cell proliferation and invasiveness were enhanced significantly, and the endocellular protein kinase B (Akt) was phosphated promptly in 5 minutes. Furthermore, we confirmed the respectively proliferative effects of CXCL12 and CXCL16 on the trophoblast. Either CXCL16 or CXCL12 stimulated the trophoblast proliferation, but CXCL16 and CXCL12 did not appear to synergism in stimulating the trophoblast proliferation.4. CXCL16 from trophoblasts was involved in the recruitment and adhesion of human first-trimester decidual immunocytes (DIC)First, we isolated human peripheral blood mononuclear cells (PBMC), and analyzed the expression of CXCR6 on CD56~+CD16~-NK cells, CD56~+CD16~+NK cells, T cells, NKT cells, y5 T cells and monocytes. Then the chemotaxis of CXCL16 to PBMC was testified by Transwell, and the chemotactic activity was quantitatively investigated by FCM. Both rhCXCL16 and trophoblast conditioned medium (TCM) exhibited chemotactic activity on peripheral blood T cells, y5 T cells and monocytes. Interestingly, the composition of the immune cells driven by the TCM and CXCL16 was quite similar to that of the decidual immunocytes in situ.Second, we isolated the decidual immunocytes for further chemotaxis. The results indicated that CXCL16 endowed the trophoblasts with the capacity to attract decidual T cells, y8 T cells and monocytes. The composition of the decidual immunocytes migrated to lower chambers was similar to that of pre-migration. Thus the first-trimester human trophoblasts produced CXCL16, which not only recruited the peripheral blood T cells, y8 T cells and monocytes to deciduas, but also attracted those cells to reside in deciduas to maintain immune milieu at the materno-fetal interface.In summary, trophoblasts express functional CXCR6/CXCL16, which increases the viability and invasiveness of trophoblasts by activation of Akt signaling pathway. The peripheral blood and decidual T cells, γδΤ cells and monocytes expressed highly CXCR6, and were recruited and resided in decidua via CXCL16/CXCR6 interaction to form the materno-fetal interface of human first-trimester pregnancy. |
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