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First published online October 6, 2003
doi: 10.1242/10.1242/dev.00729

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Trophoblasts acquire a chemokine receptor, CCR1, as they differentiate towards invasive phenotype

Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan

View larger version (56K): [in a new window]   Fig. 9. Effect of hypoxia and decidua-derived soluble factors on trophoblastic expression of CCR1. (A-D) Chorionic villi (8-weeks gestation) were cultured for 48 hours in either 20% O2 (A,B) or 1% O2 (C,D), followed by immunostaining with anti-CCR1 mAb. Explanted villous tips are traced with broken lines. In 20% O2, round cells grew out from the explanted villous tips to form a cell sheet, from which spindle-shaped cells migrated (A). CCR1 expression is detected clearly on these cells (B). In 1% O2, the individual outgrown cells are larger and the intercellular spaces are wider (C) and CCR1 expression is weak (D). (E) EVTs that outgrew in 20% O2 were isolated and cultured for a further 24 hours in the presence of decidua-conditioned medium (either with or without heat-inactivation). These EVTs were treated with either anti-CCR1 mAb or isotype-matched control mAb and analyzed by FACScalibur. There is a distinct shift in the fluorescence intensity of the cells treated with intact decidua-conditioned medium compared to those treated with heat-inactivated decidua-conditioned medium. Scale bars: 200 µm.

View larger version (81K): [in a new window]   Fig. 1. Expression of CCR1 protein and mRNA on human EVTs at 9-weeks gestation. (A-E) Serial sections of placental tissue from therapeutic hysterectomy at 9-weeks gestation were doubled-immunostained with anti-CCR1 mAb followed by rhodamine-conjugated secondary antibody plus either FITC-conjugated anti-cytokeratin 7 mAb or FITC-conjugated anti-von Willebrand factor pAb to visualize trophoblasts (A-D) and blood vessels (E), respectively. (C) is a higher magnification of the area indicated in (B). (D,E) The maternal arterial wall is traced with a dashed line. EVTs locating from the cell column (Column) through the trophoblastic shell (Shell) (A,B) and from the trophoblastic shell into the maternal artery (D,E) are shown. CCR1 expression is detected clearly on EVTs in the cell column (B,C) and trophoblastic shell (B), but is scarcely detected on cytotrophoblasts (CT) and syncytiotrophoblast (ST) (C). CCR1 expression is diminished on interstitial trophoblasts (Int TB) (B,C), whereas it is maintained on endovascular trophoblasts (Endov TB) that migrate from the trophoblastic shell into the maternal artery (E). (F) 35-cycle PCR detects a specific band that corresponds to CCR1 in cDNA derived from the microdissected cell columns at 9 weeks of gestation. No band is observed in the negative control in which total RNA from the cell columns was not reverse-transcribed. AV, anchoring villus; IVS, intervillous space; Gl, decidual gland. Scale bars: 200 µm.

View larger version (66K): [in a new window]   Fig. 2. Expression of CCR1 protein on human EVTs in the cell column at 10-weeks gestation. To determine the differentiation stage of EVTs that express CCR1 in the cell column, serial sections of placental tissue from therapeutic hysterectomy at 10-weeks gestation were either single-immunostained with anti-integrin 1 mAb followed by FITC-conjugated secondary antibody (B) or double-immunostained with anti-CCR1 mAb followed by rhodamine-conjugated secondary antibody plus FITC-conjugated anti-integrin 5 mAb (C-E). Trophoblasts are visualized by staining with anti-cytokeratin 7 mAb (A). The cytotrophoblast layer is traced with a dashed line. Two adjacent cell columns (Column 1 and Column 2) are shown. In these cell columns, the initial expression sites of CCR1 (D, arrowheads) are similar to those of integrin 5 (C,E). Note that CCR1 expression is diminished on migrating interstitial trophoblasts (Int TB) (D,E) that express both integrin 1 (B) and integrin 5 (C,E). AV, anchoring villus; Gl, decidual gland. Scale bars: 100 µm.

View larger version (97K): [in a new window]   Fig. 3. Expression of CCR1 protein on human endovascular trophoblasts at 10-weeks gestation. Serial sections of placental tissue from therapeutic hysterectomy at 10-weeks gestation were double-immunostained with anti-CCR1 mAb followed by rhodamine-conjugated secondary antibody plus either FITC-conjugated anti-cytokeratin 7 mAb or FITC-conjugated anti-von Willebrand factor pAb to visualize trophoblasts (B,E) and blood vessels (C,F), respectively. Endovascular trophoblasts (Endov TB) near the intervillous space (IVS) (A-C) and those in the deep portion of the decidual tissue (D-F) are shown. Expression of CCR1 is detected on endovascular trophoblasts that have just entered the maternal artery from the trophoblastic shell (Shell) (C), whereas it is hardly detected on the endovascular trophoblasts that reside in the maternal arteries located in the deep portion (F). Gl, decidual gland. Scale bars: 200 µm.

View larger version (37K): [in a new window]   Fig. 4. Differential expression of chemokine receptor mRNAs in the isolated human EVTs. mRNA was extracted from EVTs isolated from explant cultures of chorionic villi at 8-weeks gestation and RT-PCR performed using specific primers against chemokine receptors CCR1-11, CXCR1-5, XCR1, CX3CR1 and S26. cDNA from PBMCs was used as a positive control (top panel). The isolated EVTs express CCR1, CCR10 and XCR1 (middle panel). The negative control (total RNA from isolated EVTs without reverse transcription) is shown in the bottom panel.

View larger version (59K): [in a new window]   Fig. 5. Expression of CCR1 protein on the surface of isolated human EVTs. (A-D) Isolated EVTs (7-weeks gestation) were cultured for 4 hours to allow attachment and then double-immunostained with anti-CCR1 mAb followed by rhodamine-conjugated secondary antibody plus either FITC-conjugated anti-cytokeratin 7 mAb (A) or FITC-conjugated anti-vimentin mAb (C). (B) is a higher magnification of the area indicated in (A). (D) is a negative control for (A) in which the anti-CCR1 mAb is replaced by isotype-matched control mAb. About 50% of cytokeratin 7-positve cells (EVTs) are positive for CCR1 (A), which is clearly located on the cell surface (B), whereas CCR1 expression is scarcely detected on the vimentin-positive cells (contaminating villous stromal cells) (C). A control mAb does not stain the surface of cytokeratin 7-positive cells (D). (E) Isolated EVTs (8-weeks gestation) were treated with either anti-CCR1 mAb or isotype-matched control mAb followed by FITC-conjugated secondary antibody and analyzed by FACScalibur. A shift in the fluorescence intensity of the cells treated with anti-CCR1 mAb is observed compared to an isotype-matched control mAb. Scale bars: 50 µm.

View larger version (48K): [in a new window]   Fig. 6. mRNA expression of CCR1 ligands in human placental tissues. Chorionic and decidual samples were obtained separately from the same cases (6-9 weeks of gestation, n=5) and used for 30-cycle RT-PCR with primers against either CCR1 ligands or S26 as a housekeeping probe. PCR products from the chorionic samples and the decidual counterparts were electrophoresed (lanes C1-C5 and D1-D5, respectively). Specific bands for RANTES, MIP-1, MCP-2 and HCC-1 are detected in all five decidual samples. These chemokines are hardly detected in their chorionic counterparts.

View larger version (81K): [in a new window]   Fig. 7. Expression of CCR1 ligands at the human feto-maternal interface at 9-weeks gestation. Serial sections of placental tissue from therapeutic hysterectomy at 9-weeks gestation were immunostained with goat anti-RANTES (B,G), anti-MCP-2 (C,H) and anti-MIP-1 pAb (E,I). (A), (D) and (F) shows HE staining of (B,C), (E) and (G,H,I), respectively. In decidual tissue, RANTES is expressed in some of the resident leukocytes (DL; B) and MCP-2 is diffusely expressed in the stromal cells (DS) (C). MIP-1 is expressed in interstitial trophoblasts (Int TB) and endovascular trophoblasts (Endov TB) (E). The expression of RANTES and MCP-2 is hardly detectable in chorionic villi (G,H). There is patchy expression of MIP-1 in syncytiotrophoblast (I). FV, floating villi. Scale bars: 100 µm.

View larger version (28K): [in a new window]   Fig. 8. Effects of CCR1 ligands on migratory activity of isolated human EVTs. Isolated EVTs (6–9-weeks gestation, n=5) were allowed to migrate through a Matrigel-coated membrane filter in the absence (control) or presence of RANTES (A-C), MIP-1 (D), MCP-2 (E) and HCC-1 (F), either with or without heat-inactivation. EVTs that reached the lower surface were visualized by staining with anti-cytokeratin 7 mAb (A,B) and counted with NIH Image 1.61. The number of EVTs that migrated in the presence of intact RANTES was significantly larger than in the presence of heat-inactivated RANTES and in the control (C). Similarly, the number of migrated EVTs increased significantly in the presence of intact MIP-1 (D), MCP-2 (E) and HCC-1 (F). Scale bars: 200 µm in A, B. Mean±s.e.m. in C-F; *, P<0.05; **, P<0.01.

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