Hum. Reprod. Advance Access published online on August 6, 2008
Human Reproduction, doi:10.1093/humrep/den308
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Chemokine CXCL12 promotes the cross-talk between trophoblasts and decidual stromal cells in human first-trimester pregnancy
1 Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, Fudan University Shanghai Medical College, Shanghai 200011, PR China 2 Department of Obstetrics and Gynecology, Zhongnan Hospital of Wuhan University, Wuhan 430071, PR China 3 Department of Obstetrics and Gynecology, Hainan Medical College Affiliated Hospital, Haikou 570102, PR China
4 Correspondence address. Tel/Fax: +86-21-63457331; E-mail: dmrlq1973{at}yahoo.com.cn (M-R.D.)/djli{at}shmu.edu.cn (D-J.L.)
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionFundingReferences |
|---|
BACKGROUND: The precise mechanisms in the materno-fetal dialogue still remain unclear. The aim of this study was to investigate the role of the chemokine CXCL12 and its receptor CXCR4 in the interaction of trophoblasts and decidual stromal cells (DSCs).
METHODS: Expression of CXCL12/CXCR4 in trophoblasts and DSCs was detected by reverse transcription–polymerase chain reaction and immunochemical staining. The secretion of CXCL12 by trophoblasts was determined by enzyme-linked immunosorbent assay. The effects of CXCL12 on the biological functions of trophoblasts and DSCs were analyzed using a cell viability assay, matrigel invasion assay and zymography. Finally, a co-culture model was established to investigate the modulation of CXCL12/CXCR4 in the interaction of trophoblasts and DSCs.
RESULTS: CXCR4 was transcribed and translated by both human trophoblasts and DSCs. Human trophoblasts secreted CXCL12 spontaneously in vitro, but DSCs did not. CXCL12 induced an apparent increase in the invasiveness of trophoblasts (P < 0.01), and up-regulated matrix metalloproteinase (MMP) 9 and MMP2 activity of both trophoblasts and DSCs (both P < 0.01) in an autocrine and paracrine manner. The invasiveness and MMP9 and MMP2 activity of trophoblasts in co-culture with DSCs increased significantly (P < 0.01), and these could be inhibited by anti-CXCR4 neutralizing antibody.
CONCLUSIONS: CXCL12 secreted by human trophoblasts enhances the coordination between trophoblasts and DSCs, via the regulation of MMP9 and MMP2, which may improve the functional materno-fetal interface.
Key words: chemokine/chemokine receptor/trophoblast cell/decidual stromal cell/materno-fetal interface
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingReferences |
|---|
One of the most fascinating properties of normal pregnancy is the delicate chorus between the fetus-derived trophoblast cells and the mother-derived cells. The cells synchronize via production of cytokines, chemokines, growth factors and hormones to establish a unique materno-fetal immune milieu that contributes to fetus survival and development in maternal uterus until parturition (Herrler et al., 2003
; Veenstra van Nieuwenhoven et al., 2003; Dimitriadis et al., 2005; Higuma-Myojo et al., 2005; Staun-Ram and Shalev, 2005). Dysfunction in interaction of trophoblasts to the mother-derived cells is highly linked with some pregnancy failures, such as miscarriage, pre-eclampsia, fetal growth restriction and so on (Hustin et al., 1990; Hill et al., 1995; Kimatrai et al., 2003; Yamada et al., 2003; Ball et al., 2006; Kadyrov et al., 2006). Therefore, the partners, mothers, as well as the embryo play roles at the materno-fetal interface.
Among the various types of mother-derived cells at the materno-fetal interface, the cells named decidual stromal cells (DSCs) are the major cellular component in human decidua, and originate from the proliferation and differentiation (decidualization) of a fibroblast-like stromal cell precursor (preDSC) detected in the endometrium (Richards et al., 1995). The DSCs are involved in a series of immune regulations such as production of cytokines as well as antigen phagocytosis and presentation (Olivares et al., 1997; Ruiz et al., 1997; Kitaya et al., 2000), especially, a main source of the specific extracellular matrix (ECM) during pregnancy (Aplin et al., 1988; Loke et al., 1989; Zhu et al., 1992), indicating that the DSCs might be an important regulator of trophoblast migration and invasion. In spite of the increasing interest in DSCs biological functions, much remains to be learned about the cross-talk between DSCs and trophoblasts.
Chemokines are a sort of small molecular cytokine that are involved in a series of physiological and pathological events, including chemotaxis, cellular proliferation, differentiation, apoptosis, angiogenesis, hematopoiesis, pro-tumor and anti-tumor activity, and inflammatory disease (Luster, 1998; Rossi and Zlotnik, 2000; Yoshie, 2000; Zlotnik and Yoshie, 2000; Mackay, 2001; Balkwill, 2004). The relationship of chemokines to pregnancy has been studied by more and more investigators (Dimitriadis et al., 2005; Hannan and Salamonsen, 2007). It has been demonstrated that the invading extravillous trophoblasts (EVTs) that eventually perform endovascular invasion express chemokine CXCL12, and CXCR4 (the receptor for CXCL12) is preferentially expressed on CD16– natural killer (NK) cell subsets derived either from the peripheral blood or the decidua (Hanna et al., 2003). Our previous study has also confirmed that the first-trimester human trophoblast cells secrete CXCL12, which not only induces trophoblast proliferation in an autocrine manner, but also recruited the CD56brightCD16– NK cells into decidua in a paracrine manner (Wu et al., 2004, 2005), which suggests that chemokine CXCL12 and its receptor CXCR4 might play an important role in both immune and non-immune functions in placenta.
To better understand the mechanisms of materno-fetal dialogue, in the present study, we explored the role of CXCL12/CXCR4 in the interaction between trophoblasts and DSCs. First, we evaluated the expression of CXCL12/CXCR4 in the first-trimester human trophoblasts and DSCs at both transcriptional and translational levels. Thereafter, the effects of CXCL12/CXCR4 on the functions of trophoblasts and DSCs were determined by cell viability assay, matrigel invasion assay and gelatin zymography. Finally, a co-culture invasion model was established to investigate the modulation of CXCL12/CXCR4 interactions in the cross-talk between trophoblasts and DSCs.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingReferences |
|---|
Human placental and decidual tissue collection
All procedures involving participants in the study were approved by the Human Research Ethics Committee of Obstetrics and Gynecology Hospital, Fudan University, and all subjects completed an informed consent for the collection of tissue samples.
Human placental and decidual tissues were from elective vaginal termination of the first-trimester pregnancies (gestational age, 6–9 week) for non-medical reasons. All the tissues were immediately collected into ice-cold Dulbecco’s modified Eagle medium (DMEM) with high D-glucose (Gibco, Grand Island, NY, USA), transported to the laboratory within 30 min after surgery, and washed in calcium- and magnesium-free Hanks balanced salt solution for trophoblast or DSC isolation.
Isolation and culture of the first-trimester human trophoblast cells
The villous tissue was digested by repeated trypsin digestions according to our previous method (Zhou et al., 2007). Briefly, the placenta was digested by 0.25% trypsin (Bio Basic Inco., BBI, Ontario, Canada) and 0.02% DNase type I (Sigma Chemicals co., St Louis, MO, USA) at 37°C with gentle agitation for 5 min. Then the digested suspension was discarded, and the residual tissue was subjected to four cycles of 10 min digestion. The cell suspensions were pooled, carefully layered over a discontinuous Percoll gradient (65% to 20%, in 5% step), and centrifuged at 1000 g for 20 min. The cells sedimenting at densities between 1.048 and 1.062 g/ml were collected, and washed with DMEM–high-glucose medium. These cells were then diluted to 5 x 105 cells/well, and maintained in DMEM–high-glucose complete medium (2 mM glutamine, 25 mM HEPES, 100 UI/ml penicillin and 100 µg/ml streptomycin), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco).
Isolation and culture of human first-trimester DSC
After blood clots had been removed, DSCs were isolated according to the method described previously with slight modifications (Montes et al., 1996). Briefly, the specimens were cut into small pieces (
l mm3) and digested in six cycles of 10–20 min using 0.25% trypsin (Bio Basic Inco.) in a shaking water bath at 37°C. The cell digest was then passed through a 38 µm gauze and purified by centrifugation through a discontinuous Percoll gradient (20–60%). Then, the cells were plated into 24-well plates and maintained in DMEM–high-glucose complete medium (2 mM glutamine, 25 mM HEPES, 100 UI/ml penicillin and 100 µg/ml streptomycin). Samples of decidua from different patients were not mixed, to avoid alterations in the DSC phenotype resulting from allogeneic reaction and secretion of cytokines by lymphocytes that initially contaminate DSC cultures.
Reverse transcription–polymerase chain reaction
The isolated trophoblast cells and DSCs were seeded at a density of 1 x 106 cells/ml in 6-cell culture plates. The plates for trophoblasts culture had been pre-coated with matrigel (BD Biosciences, The Oak Park, Bedford, MA 01730, USA). After 70–80% confluence of the cells, total cellular RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA (4 µg) was used for first-strand complementary DNA (cDNA) synthesis in 20 µl reaction volume with 200 units Moloney murine leukemia virus reverse transcriptase. Then, the cDNA (10 µl) was amplified by PCR reaction in a final volume of 50 µl containing 2 mM dNTP, 0.8 µM specific primers, 1.25 U Taq DNA polymerase, 1 mM MgCl2 and 1x reaction buffer. A 5 min pre-cycle at 95°C was followed by 35 cycles of 1 min at 94°C, 30 s at 55°C and 30 s at 72°C. After the final cycle, the samples were kept at 72°C for 15 min to complete the synthesis. The primer pairs for cDNA amplification were as follows: 5'-GAA CTT CCT ATG CAA GGC AGT CC-3' (forward) and 5'-CCA TGA TGT GCT GAA ACT GGA AC-3' (reverse) for human CXCR4; 5'-ATG AAC GCC AAG GTC GTG GTC G-3' (forward) and 5'-TGT TGT TGT TCT TCA GCC G -3' (reverse) for human CXCL12; 5'-GGG GAG CCA AAA GGG TCA TCA TCT-3' (forward) and 5'-GAG GGG CCA TCC ACA GTC TTC T-3' (reverse) for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Wu et al., 2004). The expected fragment lengths of CXCR4, CXCL12 and GAPDH were 302, 202 and 235 bp, respectively. The PCR reaction products (10 µl) were electrophoresed on 2% agarose gels and ethidium bromide-stained bands were photographed, and analyzed by gel imaging systems. The relative intensity of CXCL12 or CXCR4= absorbance value of the target fragment/absorbance of GAPDH. The samples were run in triplicate, and experiments were repeated three times.
Immunohistochemistry
The placental or decidual tissues were routinely fixed with formalin and embedded in paraffin. Antigen retrieval was performed by microwave heating in 0.01 M sodium citrate buffer (pH 6.0). H2O2 (0.3%) in phosphate-buffered saline (PBS) was employed to block endogenous peroxidase activity in the sections. After treating with protein blocking solution containing 7% horse serum to block non-specific binding, the sections were incubated overnight at 4°C with mouse anti-human CXCR4 (25 µg/ml) monoclonal antibody, mouse anti-human CXCL12 (10 µg/ml) monoclonal antibody (R&D Systems, Abingdon, UK), mouse anti-human HLA-G (10 µg/ml) monoclonal antibody (Abcam, Cambridge, UK) or mouse isotype immunoglobulin (Ig) G (20 µg/ml) (Sino-America Co. Ltd, Shanghai, China). A streptavidin/biotin detection reagent kit (Beijing Zhongshan Golden Bridge Biotechnology Company, Beijing, China) with 3,3'diaminobenzidine tetrahydrochloride (DAB) was employed for signal detection, and Harris hematoxylin was used as a counter-stain. The experiments were performed with five placenta and decidua samples.
Immunocytochemistry
The isolated trophoblasts and DSCs were cultured for 24–72 h, and then fixed in 4% polyformalin and washed in PBS. After treating with 0.3% H2O2 in PBS to block endogenous peroxidase activity, the cells were blocked with 7% horse serum in PBS and incubated overnight at 4°C with anti-human cytokeratin-7 (CK7) monoclonal antibody (Zymed Laboratories, USA), anti-human vimentin monoclonal antibody, anti-human factor VIII polyclonal antibody (Sino-America Co. Ltd), mouse anti-human CXCR4 (25 µg/ml) antibody, mouse anti-human CXCL12 (10 µg/ml) antibody (R&D Systems) or mouse isotype IgG (20 µg/ml) (Sino-America Co. Ltd). CK7 and vimentin were employed as markers to identify the purity of trophoblasts. CK7, factor VIII and vimentin were markers to identify the purity of DSCs. The other procedures were as described above for immunohistochemistry. The experiments were performed with three different cell preparations.
Enzyme-linked immunosorbent assay
The purified trophoblasts were seeded in a 24-well plate pre-coated with matrigel at a density of 5 x 106 cells/ml. The trophoblast supernatants were collected at 12, 24, 36, 48, 60 and 72 h of culture. Each supernatant was centrifuged at 2000g and stored at –70°C. Human CXCL12 enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems) was used to measure chemokine production in each supernatant according to the manufacturer’s instructions. The CXCL12 assay demonstrated a sensitivity of 18 pg/ml and an intra-assay coefficient of variation of 3.4–3.9%. The ELISA assay was carried out in duplicate, in three separate experiments.
Preparation of trophoblast-conditioned medium
The freshly isolated trophoblasts were seeded at a density of 1x106 cell/ml per well in 6-well plates pre-coated with matrigel, and cultured continuously for 72 h. The supernatants, namely trophoblast-conditioned medium (TCM), were collected and centrifuged at 2000g, then stored at –70°C. The supernatants from culture medium without trophoblasts were collected as control.
Cell viability assay
The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma Chemicals] assay was applied to evaluate the effects of CXCL12 on cell viability. The isolated DSCs were re-suspended in DMEM with 10% FBS, and seeded at a density of 2 x 104 cells/well in 96-well flat-bottom microplates. After 70–80% confluence, the cells were starved with DMEM containing 1% FBS for 12 h before treatment. The medium was removed once again, and the cells were stimulated with recombinant human (rh) CXCL12 (at 0, 10, 20, 30, 50, 100, 200, 300, 400 and 500 ng/ml) or TCM. Before treatment with rhCXCL12 (100 ng/ml) or TCM, the cells in some of the wells were pre-treated with CXCL12 (40 µg/ml) or CXCR4 (20 µg/ml) neutralizing antibody (R&D Systems). In addition, mouse isotype (20 µg ml) (Sino-America Co. Ltd) was added to some wells as negative control.
MTT reagent (20 µl) was added to each well of 96-well microplates and incubated at 37°C for 4 h. The medium was decanted, and 100 µl of ethanol was added to solubilize the reactive crystals. Absorbency was measured at a wavelength of 570 nm on an automatic microplate reader. The samples were run in triplicate and experiments were repeated four times.
Matrigel invasion assay
The invasion of trophoblast cells across matrigel was evaluated objectively in an invasion chamber based on our previous procedure (Zhou et al., 2007). The dose of CXCL12 and the concentrations of neutralizing antibodies were chosen according to our previous study (Wu et al., 2004, 2005; Huang et al., 2006). The cell culture inserts (8 µm pore size, 6.5 mm diameter; Corning, Corning, NY, USA) coated with 5 µl pure matrigel were placed in a 24-well plate and two sets of invasion assays were performed as follows.
First, the purified trophoblast cells (2 x 105 in 200 µl DMEM with 1% FBS) were plated in the upper chamber and treated with vehicle, rhCXCL12 (100 ng/ml) or TCM. Before treatment with rhCXCL12 (100 ng/ml) or TCM, the cells in wells were pre-incubated with CXCL12 (40 µg/ml) or CXCR4 (20 µg/ml) neutralizing antibody (R&D Systems). The CXCL12 (40 µg/ml) or CXCR4 (20 µg/ml) neutralizing antibodies alone were used as control. The lower chamber was filled with 800 µl DMEM with 10% FBS. The cells were then incubated at 37°C for 48 h.
Second, the co-culture invasion model of trophoblasts and DSCs was established to observe the invasiveness of trophoblasts. Briefly, the isolated DSCs were seeded on the opposite side of the inserts at a density of 1 x 105/well, and cultured in DMEM with 10% FBS for 48 h. Then, the freshly isolated trophoblasts (1 x 105 in 200 µl DMEM with 1% FBS) were added to the upper surface of the upper chamber, and incubated at 37°C for 48 h. Before co-culture, the DSCs or trophoblast cells in wells were pre-treated with CXCR4 (20 µg/ml) neutralizing antibody for 30 min. Cultures of trophoblasts alone were used as control.
The inserts were removed, washed in PBS and the non-invading cells together with the Matrigel were removed from the upper surface of the filter by wiping with a cotton bud. The inserts were then fixed in methanol for 10 min at room temperature. For trophoblast culture alone, the inserts were stained directly with hematoxylin. For co-culture, the inserts were first incubated with anti-human CK7 monoclonal antibody to identify trophoblast cells (Zymed Laboratories), and then counter-stained with hematoxylin to show DSCs. The result was observed under Olympus BX51+DP70 fluorescence microscope (Olympus, Tokyo, Japan). The cells which had migrated to the lower surface were counted at a magnification of x200. Each experiment was carried out in triplicate, and repeated three times.
Gelatin zymography
The collection of culture medium for analysis by zymography was divided into two steps. First, DSCs (5 x 105 cell/ml) were seeded on the 24-well plates, and treated with various concentrations of rhCXCL12 (0, 50, 100 and 200 ng/ml), rhCXCL12 (200 ng/ml) combined with CXCL12 (40 µg/ml) neutralizing antibody or rhCXCL12 (200 ng/ml) combined with CXCR4 (20 µg/ml) neutralizing antibody. After incubation for 48 h, the supernatants were collected, and stored at –70°C. Second, the culture medium of the invasion assay was also harvested, and stored at –70°C for zymography.
The proteolytic activity of both matrix metalloproteinase 9 and 2 (MMP9 and MMP2) was measured by the technique of gelatin zymography described previously (Zhou et al., 2007). Briefly, the collected culture supernatants containing 10 µg of total protein were mixed with sodium dodecyl sulfate (SDS) loading buffer and electrophoresed on 10% SDS-polyacrylamide gels copolymerized with 0.2% gelatin. After electrophoresis, the gel was rinsed in 2.5% Triton-X 100 for 1 h to remove SDS. The gel was incubated for 12 h at 37°C in 50 mM Tris–HCl (pH 7.5), 200 mM NaCl, 10 mM CaCl2 and stained with 2.5% Coomassie Blue R250 (Sigma Chemical Co.) dissolved in 40% (v/v) methanol and 10% acetic acid. The gels were then rinsed in three different decolorant solutions (A: 30% methanol, 10% acetic acid; B: 20% methanol, 10% acetic acid; C: 10% methanol, 5% acetic acid), respectively. The gel was photographed and assayed by the Odyssey Infrared Imaging System. The gelatinolytic activity was visualized as a clear white band against a dark background of stained gelatin. The samples were run in triplicate, and experiments were repeated three times.
Statistics
The post hoc Dunnett’s t-test was employed to compare the significance levels between control and different treatment groups. Unless stated otherwise, all data are presented as mean ± SE. The differences were accepted as significant at P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingReferences |
|---|
Immunocytochemical characterization for purity of trophoblasts and DSCs
In the present study, the trophoblasts were seeded on matrigel-coated culture dishes, which made them aggregate, migrate and invade the matrigel but not fuse. The trophoblast cells were almost all stained for CK7, whereas few cells were found stained with anti-vimentin antibody (Fig. 1A and B). We observed that purity of the isolated trophoblasts was >95%.
|
The primary DSCs were polygon cells with abundant cytoplasm. They had a strong proliferative ability and could survive for more than 2 weeks in vitro. The purity of the DSCs was determined by immunocytochemistry for vimentin, cytokeratin and factor VIII. As defined by these criteria, the primary DSCs used in the present study contained <1% contaminating cells (Fig. 1D–F).
The expression of CXCL12 and CXCR4 in the first-trimester human trophoblasts
The expression of CXCL12 and CXCR4 in human trophoblasts was determined by reverse transcription–polymerase chain reaction (RT–PCR) and immunochemical staining. It was shown in Fig. 2A that the first-trimester human trophoblast cells transcribed both CXCL12 and CXCR4, and the relative intensities for CXCL12 and CXCR4 were 0.6323 ± 0.113 and 0.8796 ± 0.137, respectively. The immunocytochemical results showed that the cytomembrane and cytoplasm of the primary trophoblasts were strongly stained for CXCR4 and CXCL12 (Fig. 2B and C). Moreover, we found by immunohistochemistry that specific brown-colored staining for CXCR4 and CXCL12 was recognized in the cytoplasm and cytomembrane of villous cytotrophoblasts, syncytiotrophoblasts and EVTs (Fig. 2E and F). To further explore the expression of CXCL12/CXCR4 in trophoblasts, we used HLA-G to mark the invading EVT in deciduas (Fig. 2K). It was found that the invaded EVT in deciduas also co-expressed CXCL12 and CXCR4 protein (Fig. 2L and M).
|
The release of soluble CXCL12 in culture supernatants of trophoblasts was examined by ELISA every 12 h. The primary-cultured trophoblasts secreted CXCL12 continuously at a constant rate, and the accumulated concentration of CXCL12 was 245.2 ± 32.2 ng/ml after culture for 72 h (Fig. 2N).
The expression of CXCR4 in the first-trimester human DSCs
To validate the transcription of CXCL12 and CXCR4 in DSCs, we analyzed the gene expression intensities by RT–PCR. It was shown that, unlike trophoblasts, the first-trimester DSCs mainly transcribed CXCR4 but not CXCL12, and the relative intensity of CXCR4 mRNA was 0.5478 ± 0.09 (Fig. 2A).
After identifying CXCR4 transcription in the first-trimester DSCs, we further analyzed the expression of CXCR4 protein in the cultured cells and decidual sections by immunocytochemistry and immunohistochemistry, respectively. The results showed that the moderate-to-strong staining for CXCR4 could be recognized in the cytoplasm and membrane of DSCs in vitro and in situ (Fig. 2I and L). Therefore, the first-trimester human DSCs expressed CXCR4, but not its ligand, CXCL12 at both transcriptional and translational level.
Effects of CXCL12/CXCR4 on DSCs viability in vitro
DSCs were treated with different concentrations of rhCXCL12 to observe the effects of CXCL12/CXCR4 on DSC viability by MTT assay. We found that various concentrations of CXCL12 showed no detectable effect on the viability of the first-trimester human DSC in vitro (Fig. 3A). Moreover, TCM from trophoblasts significantly promoted the viability of DSCs, but CXCR4 or CXCL12 neutralizing antibody failed to block the increased DSCs viability induced by TCM (Fig. 3B), which suggests that TCM from trophoblasts promoted the viability of DSCs independent of CXCL12/CXCR4 interaction.
|
CXCL12 increased MMP9 and MMP2 activity of the first-trimester human DSCs
Exogenous CXCL12 was administrated to stimulate the DSCs in vitro and the proteolytic activity of MMP9 and MMP2 was measured by gelatin zymography. As shown in Fig. 4A, different concentrations of rhCXCL12 substantially increased the activity of MMP9 (92 kD) and MMP2 (72 kD) in human first-trimester DSCs in a dose-responsive manner. The gelatinolytic activity was significantly higher in 100 and 200 ng/ml CXCL12, compared with the vehicle controls, and was highest in 200 ng/ml CXCL12. Moreover, CXCL12 (40 µg/ml) or CXCR4 (20 µg/ml) neutralizing antibody completely abolished the increased activity of MMP9 and MMP2 in DSCs induced by 200 ng/ml rhCXCL12, further confirming the CXCL12/CXCR4 signaling pathway is involved in the modulation of MMP9 and MMP2 activity in DSCs (Fig. 4B).
|
CXCL12 induced invasion as well as MMP9 and MMP2 activity of the first-trimester human trophoblasts
Matrigel invasion assay was performed to evaluate the role of CXCL12 in trophoblast invasiveness. As shown in Fig. 5A, rhCXCL12 significantly promoted the invasiveness of trophoblastic cells in vitro, and this stimulatory effect was
1.7-fold higher at 100 ng/ml of CXCL12. The action of CXCL12 on trophoblast invasiveness was further demonstrated by a complete inhibition of CXCL12-stimulated trophoblast invasion by anti-CXCR4 and anti-CXCL12 neutralizing antibody. Furthermore, anti-CXCR4 and anti-CXCL12 neutralizing antibody was able to block the increased invasion of trophoblasts induced by TCM (Fig. 5E). To further explore whether CXCL12 has an autocrine effect on the invasion of trophoblasts, we also performed the invasion assay just in the presence of CXCR4 or CXCL12 neutralizing antibodies. Compared with the vehicle control, the invasiveness of trophoblasts significantly decreased in the presence of neutralizing antibody (Fig. 5C).
|
The invasiveness of trophoblasts is facilitated by degradation of the ECM of the decidua via various proteinases, among which MMP9 and MMP2 play an important role (Shimonovitz et al., 1994
; Staun-Ram et al., 2004). Therefore, culture supernatant of the upper chamber in the invasion assay was harvested, and the proteolytic activity of MMP9 and MMP2 was measured by zymography. Consistent with the invasiveness, the activity of MMP9 and MMP2 to degrade gelatin also increased correspondingly after treatment with CXCL12, compared with the control. Moreover, the neutralizing antibody to CXCR4 and CXCL12 completely inhibited the CXCL12-induced MMP9 and MMP2 proteolytic activity (Fig. 5B). The MMP9 and MMP2 activity with neutralizing antibody alone was also significantly lower than that of the control (Fig. 5D). To further elucidate the autocrine role of CXCL12/CXCR4 in trophoblast invasion, we also stimulated trophoblasts with TCM. As shown in Fig. 5E, the invasiveness of trophoblasts significantly increased after TCM treatment, and the invasive index was >2-fold of the control, but the neutralizing antibody to CXCR4 and CXCL12 only partly inhibited the TCM-induced trophoblast invasion (P < 0.01, compared with the control).
CXCL12 modulation of the interaction of human first-trimester trophoblasts with DSCs
Cell encircled with a distinct basement membrane is one of the fascinating characteristics of mature DSCs (Wewer et al., 1985), which suggests trophoblast cells interact with DSCs through the ECM. Thus, to better mimic the conditions in vivo at the materno-fetal interface, we set-up a co-culture model of trophoblasts and DSCs, in which DSCs and trophoblasts were seeded on the opposite and top side of the inserts, respectively (Fig. 6A). CK-7 was used as the marker for identifying trophoblasts, to differentiate them from DSCs that were negative for CK7. It was shown in Fig. 6B that the number of migrated trophoblasts in the co-culture was significantly more than the cultured trophoblasts alone (P < 0.01). When DSCs or trophoblasts in the co-culture were pre-treated with anti-CXCR4 blocking antibody, the invasiveness of trophoblasts decreased significantly (P < 0.01, compared with the non-treatment), but was still stronger than the cultured trophoblast alone (P < 0.01). Although no obvious difference in trophoblast invasion was found between the pre-treated DSC and pre-treated trophoblast in co-culture, the invaded trophoblasts cells seemed to be less when trophoblasts were pre-blocked with anti-CXCR4 neutralizing antibody.
|
To explore the possible down-stream molecules of CXCL12/CXCR4 responsible for the increased invasiveness of trophoblasts in the co-culture model, we also collected the culture medium from the co-culture model and measured the proteolytic activity of MMP9 and MMP2 by zymography. As shown in Fig. 6C, the activity of both MMP9 and MMP2 significantly increased in the co-culture model and was
2.4-fold and 3.2-fold higher, respectively, compared with the cultured trophoblasts alone. Moreover, treatment with anti-CXCR4 neutralizing antibody to trophoblasts or DSCs significantly inhibited the increased activity of MMP9 and MMP2 in the co-culture (P < 0.01, compared with the non-treatment), but this inhibition by anti-CXCR4 blocking antibody was incomplete (P < 0.01, compared with the cultured trophoblasts alone). |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingReferences |
|---|
Among various chemokines and their receptors, CXCL12/CXCR4 is an extraordinary chemokine/chemokine receptor pair, and appears unique in its biological functions. Apart from the important impact on human immunodeficiency virus-1 infection, CXCR4 and its specific ligand CXCL12 play a key role in lymphocyte trafficking, cellular proliferation, organogenesis, vascularization and embryogenesis (Murdoch, 2000
; Zhu et al., 2002; Wu et al., 2004, 2005). It has been also found that CXCL12/CXCR4 is involved in the invasion of some malignant cells (Müller et al., 2001; Samara et al., 2004). In the present study, we have confirmed the expression of CXCL12/CXCR4 in the first-trimester human trophoblasts and the expression of CXCR4 in the first-trimester human DSCs at both transcriptional and translational level.
As a key component of human placenta, trophoblast is the only embryo-derived cell which interacts with the maternal-derived cells directly. Trophoblast proliferation and invasion are a series of tightly controlled processes that are pivotal to implantation and placentation. Excessive or insufficient proliferation and invasion of trophoblast has been confirmed as highly related to some pregnancy failures (Torry et al., 1998; Kaufmann et al., 2003; Bose et al., 2006). Our previous study has demonstrated that CXCL12 secreted by trophoblast cells induced their proliferation via binding to CXCR4 expressed by the same cells (Wu et al., 2004). In the present study, we found that CXCL12/CXCR4 interaction in trophoblasts also promotes the invasiveness as well as MMP9 and MMP2 activity of these cells. It is reasonable to conclude that the coexpression of CXCL12/CXCR4 contributes to the regulation of trophoblast function in an autocrine manner, through which trophoblast cells promote not only their own proliferation, but also migration and invasion via modulating the activity of MMP9 and MMP2. However, an addition of anti-CXCR4 and anti-CXCL12 neutralizing antibody only partly blocked the stimulatory effect of TCM on trophoblast invasion, suggesting other signals or molecules are also involved in the TCM-induced action, besides CXCL12/CXCR4 signaling pathway.
When trophoblasts invade deep into the deciduas, they are in intimate contact with DSCs, another important constituent at the materno-fetal interface. It has been shown that EVT in a cytotrophoblast column, and DSCs could attach to each other by desmosomal junction (Babawale et al., 2002), suggesting that there are communications between them, but the detailed mechanisms remain unclear. In our present study, the first-trimester human DSCs were confirmed to express CXCR4, whereas the ligand for CXCR4 and CXCL12 was mainly produced by trophoblasts. We propose that trophoblasts might cross-talk with DSCs via CXCL12/CXCR4 signaling pathway.
To verify our speculation, we first observed the effect of CXCL12 on the viability of DSCs in vitro. However, unlike trophoblasts that are able to proliferate by CXCL12 stimulation (Wu et al., 2004), exogenous CXCL12 exhibited no detectable effect on the viability of DSCs. Moreover, neutralizing antibody to CXCR4 and CXCL12 failed to block the increased viability of DSCs induced by the TCM, suggesting trophoblast has the potential to modulate the viability of DSCs, but this action was independent of the CXCL12/CXCR4 pathway.
However, it was found in the present study that exogenous CXCL12 increased the MMP9 and MMP2 activity by DSCs in a dose-responsive manner, and the specificity of CXCL12 action was further demonstrated by a complete inhibition of the CXCL12-stimulated DSCs MMP activity by CXCR4 and CXCL12 neutralizing antibody. Furthermore, CXCL12 also promoted MMP9 and MMP2 activity of trophoblasts in an autocrine manner. MMP9 and MMP2 have been shown to be critical determinants for trophoblast migration and invasion (Shimonovitz et al., 1994; Staun-Ram et al., 2004). Thus, it may be speculated that CXCL12/CXCR4 interaction promotes the MMP activity by both trophoblasts and DSCs, which contributes to the coordination of the two cell types, and further leads to trophoblast migration and invasion.
To better understand the role of CXCL12/CXCR4 interaction in the cross-talk between trophoblasts and DSCs, we established a co-culture model. As we predicted, after co-culture with DSCs, the invasiveness of trophoblasts significantly increased, and the MMP9 and MMP2 activity in the co-culture model was also up-regulated correspondingly. Furthermore, when trophoblasts in the cocluture were pre-treated with CXCR4 neutralizing antibody, both invasiveness and MMP activity of trophoblasts significantly decreased. In our experiments, DSCs were not a source of CXCL12, but we also demonstrated in the present study that trophoblasts are capable of secreting CXCL12 spontaneously in vitro and that CXCL12 can increase MMP activity of DSCs. Thus, we also pre-treated DSCs with CXCR4 neutralizing antibody in the co-culture model to explore whether the effects of DSCs on trophoblast invasion could be mediated by trophoblast-derived CXCL12 in a paracine manner. It was clearly shown that both the MMP activity and trophoblast invasion significantly decreased in co-culture with the pre-treated DSCs. Therefore, on the one hand trophoblasts modulate their own functions, by coexpression of CXCL12/CXCR4, in an autocrine manner and on the other they communicate with CXCR4- positive DSCs via secretion of CXCL12 in a paracrine manner, which is beneficial to the MMP activity, ECM degradation and invasion of trophoblasts. In addition, CXCR4 blocking antibody only partly inhibited the invasion and MMP activity in the co-culture model, suggesting the communication between trophoblasts and DSCs is complicated, and other molecules or signals are involved in this event.
In fact, DSCs are also capable of producing some inhibitory factors for trophoblast invasion (Staun-Ram and Shalev, 2005). However, in our co-culture model, the pre-seeded DSCs could degrade the matrigel via production of MMP9 and MMP2, and promote the invasion of trophoblasts. Therefore, there might be a precise modulation of DSCs on trophoblasts invasion, not merely inhibition or promotion, which contributes to the appropriate and limited invasion of trophoblasts.
Our previous study demonstrated that first-trimester human cytotrophoblasts co-expressed CXCL16 and CXCR6 and secreted CXCL16, which induced their proliferation and invasion in an autocrine manner (Huang et al., 2006). Moreover, first-trimester human DSCs co-expressed CCL2 and CCR2 and secreted CCL2 spontaneously (He et al., 2007). Therefore, there might be a complicated chemokine network at the materno-fetal interface. Trophoblasts or DSCs not only modulate their own biological functions via their respective chemokines/chemokine receptors, but also interact with each other through secretion of chemokines, through which trophobalsts and DSCs build a multiple connection, and participate in the complex materno-fetal immune regulation.
In summary, our study has demonstrated that first-trimester human trophoblasts promote their own invasiveness and MMP activity, through coexpression of CXCL12 and CXCR4, in an autocrine manner. Moreover, CXCL12 secreted by trophoblasts enhances the coordination between trophoblasts and DSCs via regulating MMP9 and MMP2 activity of both in an autocrine and paracrine manner, which might be one of the mechanisms by which trophoblasts and DSCs communicate reciprocally, and further modulate the trophoblast invasion.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingReferences |
|---|
National Basic Research Program of China (2006CB944007) to D-J.L; Key Project of National Natural Science Foundation of China (30730087) to D-J.L.; National Natural Science Foundation of China (30670787) to D-J.L.; Foundation for Younger Investigator of Chinese Education Ministry (20070246037) to M-R.D.; Natural Science Foundation of Shanghai (06ZR14120) to M-R.D.; Shanghai Leading Academic Discipline Project B117 to D-J.L.; Program for Outstanding Medical Academic Leader to D-J.L.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingReferences |
|---|
Aplin JD, Charlton AK, Ayad S. An immunohistochemical study of human endometrial extracellular matrix during the menstrual cycle and first trimester of pregnancy. Cell Tissue Res (1988) 253:231–240.[Web of Science][Medline]
Babawale MO, Mobberley MA, Ryder TA, Elder MG, Sullivan MH. Ultrastructure of the early human feto-maternal interface co-cultured in vitro. Hum Reprod (2002) 17:1351–1357.
Balkwill F. Cancer and the chemokine network. Nat Rev Cancer (2004) 4:540–550.[CrossRef][Web of Science][Medline]
Ball E, Bulmer JN, Ayis S, Lyall F, Robson SC. Late sporadic miscarriage is associated with abnormalities in spiral artery transformation and trophoblast invasion. J Pathol (2006) 208:535–542.[CrossRef][Web of Science][Medline]
Bose P, Kadyrov M, Goldin R, Hahn S, Backos M, Regan L, Huppertz B. Aberrations of early trophoblast differentiation predispose to pregnancy failure: lessons from the anti-phospholipid syndrome. Placenta (2006) 27:869–875.[CrossRef][Web of Science][Medline]
Dimitriadis E, White CA, Jones RL, Salamonsen LA. Cytokines, chemokines and growth factors in endometrium related to implantation. Hum Reprod Update (2005) 11:613–630.
Hanna J, Wald O, Goldman-Wohl D, Prus D, Markel G, Gazit R, Katz G, Haimov-Kochman R, Fujii N, Yagel S, et al. CXCL12 expression by invasive trophoblasts induces the specific migration of CD16- human natural killer cells. Blood (2003) 102:1569–1577.
Hannan NJ, Salamonsen LA. Role of chemokines in the endometrium and in embryo implantation. Curr Opin Obstet Gynecol (2007) 19:266–272.[Web of Science][Medline]
He YY, Du MR, Guo PF, He XJ, Zhou WH, Zhu XY, Li DJ. Regulation of C-C motif chemokine ligand 2 and its receptor in human decidual stromal cells by pregnancy-associated hormones in early gestation. Hum Reprod (2007) 22:2733–2742.
Herrler A, von Rango U, Beier HM. Embryo-maternal signalling: how the embryo starts talking to its mother to accomplish implantation. Reprod Biomed Online (2003) 6:244–256.[Medline]
Higuma-Myojo S, Sasaki Y, Miyazaki S, Sakai M, Siozaki A, Miwa N Saito S. Cytokine profile of natural killer cells in early human pregnancy. Am J Reprod Immunol (2005) 54:21–29.[Medline]
Hill JA, Polgar K, Anderson DJ. T-helper 1-type immunity to trophoblast in women with recurrent spontaneous abortion. JAMA (1995) 273:1933–1936.
Huang Y, Zhu XY, Du MR, Wu X, Wang MY, Li DJ. Chemokine CXCL16, a scavenger receptor, induces proliferation and invasion of first-trimester human trophoblast cells in an autocrine manner. Hum Reprod (2006) 21:1083–1091.
Hustin J, Jauniaux E, Schaaps JP. Histological study of the materno-embryonic interface in spontaneous abortion. Placenta (1990) 11:477–486.[CrossRef][Web of Science][Medline]
Kadyrov M, Kingdom JC, Huppertz B. Divergent trophoblast invasion and apoptosis in placental bed spiral arteries from pregnancies complicated by maternal anemia and early-onset preeclampsia/intrauterine growth restriction. Am J Obstet Gynecol (2006) 194:557–563.[CrossRef][Web of Science][Medline]
Kaufmann P, Black S, Huppertz B. Endovascular trophoblast invasion: implications for the pathogenesis of intrauterine growth retardation and preeclampsia. Biol Reprod (2003) 69:1–7.
Kimatrai M, Oliver C, Abadia-Molina AC, Garcia-Pacheco JM, Olivares EG. Contractile activity of human decidual stromal cells. J Clin Endocrinol Metab (2003) 88:844–849.
Kitaya K, Yasuda J, Yagi I, Tada Y, Fushiki S, Honjo H. IL-15 expression at human endometrium and decidua. Biol Reprod (2000) 63:683–687.
Loke YW, Gardner L, Burland K, King A. Laminin in human trophoblast–decidua interaction. Hum Reprod (1989) 4:457–463.
Luster AD. Chemokines-chemotactic cytokines that mediate inflammation. N Engl J Med (1998) 338:436–445.
Mackay CR. Chemokines: immunology’s high impact factors. Nat Immunol (2001) 2:95–101.[CrossRef][Web of Science][Medline]
Montes MJ, Aleman P, Garcia-Tortosa C, Borja C, Ruiz C, Garcia-Olivares E. Cultured human decidual stromal cells express antigens associated with hematopoietic cells. J Reprod Immunol (1996) 30:53–66.[CrossRef][Web of Science][Medline]
Müller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, McClanahan T, Murphy E, Yuan W, Wagner SN, et al. Involvement of chemokine receptors in breast cancer metastasis. Nature (2001) 410:50–56.[CrossRef][Web of Science][Medline]
Murdoch C. CXCR4: chemokine receptor extraordinaire. Immunol Rev (2000) 177:175–184.[CrossRef][Web of Science][Medline]
Olivares EG, Montes MJ, Oliver C, Galindo JA, Ruiz C. Cultured human decidual stromal cells express B7-1 (CD80) and B7-2 (CD86) and stimulate allogeneic T cells. Biol Reprod (1997) 57:609–615.[Abstract]
Richards RG, Brar AK, Frank GR, Hartman SM, Jikihara H. Fibroblast cells from term human decidua closely resemble endometrial stromal cells: induction of prolactin and insulin-like growth factor binding protein-1 expression. Biol Reprod (1995) 52:609–615.[Abstract]
Rossi D, Zlotnik A. The biology of chemokines and their receptors. Annu Rev Immunol (2000) 18:217–242.[CrossRef][Web of Science][Medline]
Ruiz C, Montes MJ, Abadia-Molina AC, Olivares EG. Phagocytosis by fresh and cultured human decidual stromal cells: opposite effects of interleukin-1 alpha and progesterone. J Reprod Immunol (1997) 33:15–26.[CrossRef][Web of Science][Medline]
Samara GJ, Lawrence DM, Chiarelli CJ, Valentino MD, Lyubsky S, Zucker S, Vaday GG. CXCR4-mediated adhesion and MMP-9 secretion in head and neck squamous cell carcinoma. Cancer Letters (2004) 214:231–241.[CrossRef][Web of Science][Medline]
Shimonovitz S, Hurwitz A, Dushnik M, Anteby E, Geva-Eldar T, Yagel S. Developmental regulation of the expression of 72 and 92kd type IV collagenases in human trophoblasts: a possible mechanism for control of trophoblast invasion. Am J Obstet Gynecol (1994) 171:832–838.[Web of Science][Medline]
Staun-Ram E, Shalev E. Human trophoblast function during the implantation process. Reprod Biol Endocrinol (2005) 3:56.[CrossRef][Medline]
Staun-Ram E, Goldman S, Gabarin D, Shalev E. Expression and importance of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) in human trophoblast invasion. Reprod Biol Endocrinol (2004) 2:59.[CrossRef][Medline]
Torry DS, Labarrere CA, McIntyre JA. Uteroplacental vascular involvement in recurrent spontaneous abortion. Curr Opin Obstet Gynecol (1998) 10:379–382.[CrossRef][Web of Science][Medline]
Veenstra van Nieuwenhoven AL, Heineman MJ, Faas MM. The immunology of successful pregnancy. Hum Reprod Update (2003) 9:347–357.
Wewer UM, Faber M, Liotta LA, Albrechtsen R. Immunochemical and ultrastructural assessment of the nature of the pericellular basement membrane of human decidual cells. Lab Invest (1985) 53:624–633.[Web of Science][Medline]
Wu X, Li DJ, Yuan MM, Zhu Y, Wang MY. The expression of CXCR4/CXCL12 in first-trimester human trophoblast cells. Biol Reprod (2004) 70:1877–1885.
Wu X, Jin LP, Yuan MM, Zhu Y, Wang MY, Li DJ. Human first-trimester trophoblast cells recruit CD56brightCD16- NK cells into decidua by way of expressing and secreting of CXCL12/stromal cell-derived factor 1. J Immunol (2005) 175:61–68.
Yamada H, Morikawa M, Kato EH, Shimada S, Kobashi G, Minakami H. Pre-conceptional natural killer cell activity and percentage as predictors of biochemical pregnancy and spontaneous abortion with normal chromosome karyotype. Am J Reprod Immunol (2003) 50:351–354.[CrossRef][Web of Science][Medline]
Yoshie O. Role of chemokines in trafficking of lymphocytes and dendritic cells. Int J Hematol (2000) 72:399–407.[Web of Science][Medline]
Zhou WH, Du MR, Dong L, Zhu XY, Yang JY, He YY, Li DJ. Cyclosporin A increases expression of matrix metalloproteinase 9 and 2 and invasiveness in vitro of the first-trimester human trophoblast cells via the mitogen-activated protein kinase pathway. Hum Reprod (2007) 22:2743–2750.
Zhu HH, Huang JR, Mazela J, Elias J, Tseng L. Progestin stimulates the biosynthesis of fibronectin and accumulation of fibronectin mRNA in human endometrial stromal cells. Hum Reprod (1992) 7:141–146.
Zhu Y, Yu T, Zhang XC, Nagasawa T, Wu JY, Rao Y. Role of the chemokine SDF-1 as the meningeal attractant for embryonic cerebellar neurons. Nat Neurosci (2002) 5:719–720.[Web of Science][Medline]
Zlotnik A, Yoshie O. Chemokines: a new classification system and their role in immunity. Immunity (2000) 12:121–127.[CrossRef][Web of Science][Medline]
Submitted on December 9, 2007; resubmitted on July 9, 2008; accepted on July 16, 2008.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||