Induction of Tumor Necrosis Factor and Interleukin-6 mRNA in Human Cytotrophoblast Cells Exposed to Lipopolysaccharide

[摘要] Objective:The cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) have previously been identified inplacental tissue and are known to be mediators of infection-associated induction of the host immune system. This studywas undertaken to better characterize the in vitro regulation of these cytokines in cytotrophoblast cells when challengedwith the bacterial product lipopolysaccharide (LPS).Methods:Term placentas were freshly collected, digested with trypsin/DNase, and subjected to Percoll gradient centrifugation to isolate cytotrophoblasts. Either immediately or after overnight incubation, LPS (1 μg/ml) or media alone was added to the cell cultures for 0, 1, 2, 4, 8, 24, 48, and 72 h. Total cellular RNA was isolated by the guanidinium thiocyanate/cesium chloride methodology. RNA samples were run on 1% agarose-formaldehyde gels and subsequently transferred to nylon filters. Blots were hybridized with the appropriate P32-radiolabeled probe.Results:In non-LPS-treated cells, minimal amounts of TNF mRNA could be detected at zero time, or throughout the incubation periods. Conversely, LPS exposure resulted in detectable signal starting at 1 h and peaking at 2 h after the addition of LPS. Overnight incubation gave stronger TNF signals in the LPS stimulated cells, although the kinetics of this response remained similar to zero time exposure. IL-6 was likewise minimally expressed at zero time, although non-stimulated cell cultures demonstrated progressive increases in mRNA expression which was maximal at 16 h after plating. LPS further augmented the transcription of IL-6 mRNA, with peak signals seen at 4 h after LPS stimulation. Again, overnight incubation of the cytotrophoblasts increased baseline and LPS-induced IL-6 mRNA responses. Long-term constant exposure of cells to LPS did not demonstrate any evidence of prolonged signaling. LPS did not alter mRNA expression of the placental gene H19 or the oncogene FOS.Conclusions:These data demonstrate the induction of TNF and IL-6 mRNA in cytotrophoblast cells with LPS.These transcriptional events are kinetically distinct and short term in nature. Overnight incubation accentuates theTNF and IL-6 mRNA signal and allows for an augmented response to LPS.

[学科分类] 妇产科学