[Expression and function of chemokine TARC/CCR4 at fetal-maternal interface in first trimester]

To investigate the expression and function of thymus and activation regulated chemokine (TARC) and its special receptor CCR4 at placenta villous in the first trimester placenta villous.

Placenta villous was collected from healthy women undergoing artificial abortion at 6 to 8 weeks of gestation. mRNA levels of TARC, CCR4 were analyzed using semi-quantitative reverse transcription (RT)-PCR methods. Immunohistochemistry assay was used to assess the protein localization and expression of TARC, CCR4. Additionally, extravillous cytotrophoblasts were isolated and cultured. Expression of TARC and CCR4 was measured by immunofluorescence assay. Invasion of cell line HTR8/SVneo was analyzed by transwell assay at concentration of 10, 25, 50 and 100 ng/ml of TARC matched with RPMI 1640 fetal bovine serum free culture medium as control group. In the mean time, blocking experiment was also added to detect TARC regulating cell invasion, which were classified into four groups: control, 100 ng/ml rhTARC, 20 µg/ml anti-TARC+100 ng/ml rhTARC, 100 ng/ml rhTARC+20 µg/ml IgG. The influence of 100 ng/ml TARC on expression level of integrin-α5 and integrin-β1 were measured by using western-blot assay.

(1) In vivo assay:expression of TARC and CCR4 mRNA were detectable in first trimester placenta villous, TARC protein was localized in cytotrophoblasts, syncytiotrophoblasts and cell column especially on the distal portion, while CCR4 protein was localized on invading interstitial cytotrophobalsts. (2) In vitro assay: a. TARC, CCR4 was also expressed in primary isolated extravillous cytotrophoblasts by immunofluorescence assay; b. Matrigel invasion assay demonstrated that TARC had specific dose dependent stimulatory effects on the cells invading through the matrigel precoated filter, the number of cells migration into the lower chamber were:142±31 at 10 ng/ml group, 161±46 at 25 ng/ml group, 201±30 at 50 ng/ml group, 312±48 at 100 ng/ml group, 117±33 at control group, the significant response observed from 25 ng/ml (P<0.05) and reached a peak effect at 100 ng/ml (P<0.01); c. Blocking experiment demonstrated that when trophoblast invasion was monitored in response to TARC neutralizing antibody (15 µg/ml) together with rhTARC 100 ng/ml. The stimulatory activity of rhTARC was completely overcome, with the cells invasion into the lower chambers were 100 ng/ml rhTARC, 20 µg/ml anti-TARC+100 ng/ml rhTARC, 100 ng/ml rhTARC+20 µg/ml IgG, control: 313±47, 113±41, 287±75 and 128±23, respectively; d. Western-blot assay demonstrated that if cells were treated with 100 ng/ml rhTARC, the expression of integrin-α5 were significantly increased (P<0.01), integrin-β1 level also increased when compared with control (P<0.05).

TARC was expressed specifically at human fetal-maternal interface. Trophoblast invasion and migration mainly was regulated by up-regulation integrin-α5 and integrin-β1, which plays an role in trophoblasts differentiation and placentation.