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发布于 2009-01-21 · 浏览 1177 · IP 山东山东
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First trimester trophoblast cells express TLRs
The first objective of this study was to determine the expression pattern of TLR-2 and TLR-4 in first trimester placental villous and extravillous tissues. As shown in Fig. 1A, positive immunoreactivity was observed for TLR-2 and TLR-4 in first trimester placental villi (i and iii) and extravillous tissues (ii and iv). Extravillous trophoblast cells displayed strong positive immunoreactivity for both TLR-2 (ii) and TLR-4 (iv). Interestingly, in the villous tissues, expression of TLR-2 (i) and TLR-4 (iii) was restricted to the villous cytotrophoblast cells, while the syncythiotrophoblast cells were negative for both receptors. To further characterize these observations, the expression of TLR-2 and TLR-4 in first trimester trophoblast primary cultures and cell lines was evaluated (Fig. 1B). Similarly, as observed in the tissue sections, first trimester trophoblast cells showed positive immunoreactivity for both TLR-2 (i and iii) and TLR-4 (ii and iv). The staining on the cell cultures was localized both intracellularly, as well as on the cell surface and displayed a polarized pattern.
To confirm the specificity of these findings, the expression of TLR-2 and TLR-4 by trophoblast cells was evaluated by Western blot analysis. As shown in Fig. 1C, primary cultures of first trimester trophoblast isolated from placental villi, as well as the first trimester trophoblast cell lines, 3A and H8, expressed a 90-kDa protein corresponding to TLR-2, and the 88-kDa TLR-4 protein. In addition, all cells expressed the 35-kDa TLR signaling adapter protein, MyD88 (Fig. 1C), suggesting that TLR-2 and TLR-4 expressed by first trimester trophoblast cells have the potential to signal and, therefore, be functional.
Because TLR-2 has been shown to co-operate with either TLR-1 or TLR-6 for ligand recognition (34–37), the expression of these receptors was determined in first trimester trophoblast cells. As shown in Fig. 1D, first trimester trophoblast cells expressed the 350-bp product for TLR-1, but failed to express the 500-bp product for TLR-6.
TLR-2, but not TLR-4, reduces trophoblast cell viability
Once the expression of TLR-2, TLR-4, and MyD88 by first trimester trophoblast cells had been established, the biological function of these receptors in trophoblast cells was evaluated. As shown in Fig. 2A, when trophoblast cells were incubated in the presence of the TLR-2 agonist, PDG, a significant decrease in cell viability was observed, as determined by the CellTiter 96 assay. Treatment of cells with the TLR-4 agonist, LPS, did not reduce trophoblast cell viability. However, after 48 h of treatment with LPS, there was a slight but significant increase in cell viability ( p 0.001), which was not evident after 72 and 96 h of treatment. In contrast, the effect of PDG on cell viability occurred in a time dependent manner, with a reduction in trophoblast viability of 25.93 2.31% after 48 h ( p 0.001), 33.75 0.65% after 72 h ( p 0.05), and 72.33 4.79% following 96 h ( p 0.001) of treatment. Although the reduction in trophoblast cell viability induced by PDG was dose-dependent (Fig. 2B), no such effect could be detected following treatment with LPS at varying concentrations (Fig. 2C). In a separate experiment, PDG (80 g/ml) reduced trophoblast cell viability by 74%, however, in the presence of a blocking anti-TLR-2 mAb (50 g/ml), PDG reduced cell viability by 66% ( p 0.05 relative to PDG alone; data not shown)
TLR-2, but not TLR-4, mediates apoptosis in first trimester trophoblast cells
The next objective was to determine whether the decrease in trophoblast viability through TLR-2 was the result of an induction in apoptosis. Therefore, first trimester trophoblast cells were incubated with either no treatment (NT), PDG, or LPS (80 g/ml). Following a 48 h treatment, the cells were double stained with propidium iodide and Hoechst 33342 dye and the number of apoptotic cells were analyzed by flow cytometry. Treatment with PDG resulted in a 61.72% increase in the number of apoptotic trophoblast cells compared with the untreated control, while no such increase in apoptotic trophoblast cells was observed following treatment with LPS (Fig. 3).
First trimester trophoblast cells express TLRs
The first objective of this study was to determine the expression pattern of TLR-2 and TLR-4 in first trimester placental villous and extravillous tissues. As shown in Fig. 1A, positive immunoreactivity was observed for TLR-2 and TLR-4 in first trimester placental villi (i and iii) and extravillous tissues (ii and iv). Extravillous trophoblast cells displayed strong positive immunoreactivity for both TLR-2 (ii) and TLR-4 (iv). Interestingly, in the villous tissues, expression of TLR-2 (i) and TLR-4 (iii) was restricted to the villous cytotrophoblast cells, while the syncythiotrophoblast cells were negative for both receptors. To further characterize these observations, the expression of TLR-2 and TLR-4 in first trimester trophoblast primary cultures and cell lines was evaluated (Fig. 1B). Similarly, as observed in the tissue sections, first trimester trophoblast cells showed positive immunoreactivity for both TLR-2 (i and iii) and TLR-4 (ii and iv). The staining on the cell cultures was localized both intracellularly, as well as on the cell surface and displayed a polarized pattern.
To confirm the specificity of these findings, the expression of TLR-2 and TLR-4 by trophoblast cells was evaluated by Western blot analysis. As shown in Fig. 1C, primary cultures of first trimester trophoblast isolated from placental villi, as well as the first trimester trophoblast cell lines, 3A and H8, expressed a 90-kDa protein corresponding to TLR-2, and the 88-kDa TLR-4 protein. In addition, all cells expressed the 35-kDa TLR signaling adapter protein, MyD88 (Fig. 1C), suggesting that TLR-2 and TLR-4 expressed by first trimester trophoblast cells have the potential to signal and, therefore, be functional.
Because TLR-2 has been shown to co-operate with either TLR-1 or TLR-6 for ligand recognition (34–37), the expression of these receptors was determined in first trimester trophoblast cells. As shown in Fig. 1D, first trimester trophoblast cells expressed the 350-bp product for TLR-1, but failed to express the 500-bp product for TLR-6.
TLR-2, but not TLR-4, reduces trophoblast cell viability
Once the expression of TLR-2, TLR-4, and MyD88 by first trimester trophoblast cells had been established, the biological function of these receptors in trophoblast cells was evaluated. As shown in Fig. 2A, when trophoblast cells were incubated in the presence of the TLR-2 agonist, PDG, a significant decrease in cell viability was observed, as determined by the CellTiter 96 assay. Treatment of cells with the TLR-4 agonist, LPS, did not reduce trophoblast cell viability. However, after 48 h of treatment with LPS, there was a slight but significant increase in cell viability ( p 0.001), which was not evident after 72 and 96 h of treatment. In contrast, the effect of PDG on cell viability occurred in a time dependent manner, with a reduction in trophoblast viability of 25.93 2.31% after 48 h ( p 0.001), 33.75 0.65% after 72 h ( p 0.05), and 72.33 4.79% following 96 h ( p 0.001) of treatment. Although the reduction in trophoblast cell viability induced by PDG was dose-dependent (Fig. 2B), no such effect could be detected following treatment with LPS at varying concentrations (Fig. 2C). In a separate experiment, PDG (80 g/ml) reduced trophoblast cell viability by 74%, however, in the presence of a blocking anti-TLR-2 mAb (50 g/ml), PDG reduced cell viability by 66% ( p 0.05 relative to PDG alone; data not shown)
TLR-2, but not TLR-4, mediates apoptosis in first trimester trophoblast cells
The next objective was to determine whether the decrease in trophoblast viability through TLR-2 was the result of an induction in apoptosis. Therefore, first trimester trophoblast cells were incubated with either no treatment (NT), PDG, or LPS (80 g/ml). Following a 48 h treatment, the cells were double stained with propidium iodide and Hoechst 33342 dye and the number of apoptotic cells were analyzed by flow cytometry. Treatment with PDG resulted in a 61.72% increase in the number of apoptotic trophoblast cells compared with the untreated control, while no such increase in apoptotic trophoblast cells was observed following treatment with LPS (Fig. 3).
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