IL-10 enhances promoter activity of ILT4 gene and up-regulates its expression in THP-1 cells

Abstract

This study examined the effect of IL-10 on immunoglobulin-like transcript (ILT4) expression of human monocytic leukemic cell line THP-1, especially the role of the ILT4 promoter activity. ILT4 promoter area was amplified by PCR, and was cloned into the eukaryotic expressing vector pGL3-Basic. The pGL3-ILTP obtained was tested by double endonuclease digestion and sequencing. Then, the recombinant plasmid was transfected into THP-1 cells by using lipofectamine. After culture with IL-10 for 12 h, the mRNA extracted from THP-1 cells was detected by RT-PCR and the protein was detected by FACS. The dual-luciferase reporter assay system was employed to detect the activity of ILT4 promoter with or without IL-10. The results showed that the activity of pGL3-ILTP was significantly increased and was more than ten times that of pGL3-Basic cells. After culture with IL-10 for 12 h, the expression of ILT4 protein and its mean fluorescence intensity (MFI) were increased. Moreover, the mRNA was remarkably higher than that of the control group. Dual-luciferase reporter assay revealed that ILT4 promoter was much more activated after being treated with IL-10. We were led to conclude that pGL3-ILTP containing ILT4 promoter was constructed successfully. The expression of ILT4 could be up-regulated by IL-10 both at the transcriptional and translational level. Furthermore, ILT4 promoter could be much more active after addition of IL-10. This study suggests that IL-10 up-regulates ILT4 expression on monocytes via increasing ILT4 gene promoter activity, which may have implication for inducing transplantation tolerance in clinical practice.

Keywords

IL-10 / ILT4 / THP-1 / immune tolerance / promoter

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