ABSTRACT
Objective To study the biological behaviors and effects of immunoglobulin-like transcript-4 (ILT4) expression in mononuclear cells on the prognosis of sepsis.Methods ILT4 +/+ (WT) and ILT4-knockout mice (ILT4-/-) male BALB/c mice were used for sepsis modeling using cecal ligation puncture (CLP).Flow cytometry was used to measure the levels of expression of ILT4 and major histocompatihility complex class Ⅱ molecules (MHC-Ⅱ) in mononuclear cells of peripheral blood 24 h after CLP.ELISA was used to measure the concentrations of interleukin-6 (IL-6) and serum tumor necrosis factor-alpha (TNF-α) in different groups of mice at 0 h,6 h,12 h,and 24 h after CLP to monitor the survival and prognosis over the course of 168 h.Results ILT4 was highly expressed in mononuclear cells of the peripheral blood of septic mice 24 h after CLP in comparison with that before CLP (1292.00 ± 143.70) vs.(193.50 ± 52.54),P ＜ 0.05.MHC-Ⅱ expression in mononuclear cells of the peripheral blood in ILT4-/-mice was significantly higher than that in WT mice (49.38 ± 5.66)％ vs.(24.25 ± 6.76) ％,P ＜ 0.05).Serum IL-6 was significantly elevated 24 h after CLP compared with that before CLP (470.75 ± 88.03) vs.(54.25 ± 20.04),P ＜ 0.05.The serum IL-6 concentration was much lower in ILT4-/-mice thanthatin MT mice (241.25 ± 45.10)vs.(470.75 ± 88.03),P ＜ 0.05;whereas,there was no significant difference in TNF-α expression between two groups of mice (50.88 ± 6.38) vs.(53.13 ± 5.49),P ＞ 0.05.The survival rate of ILT4-/-mice was significantly higher after CLP compared with WT mice (P ＜ 0.05).Conclusion The high level of ILT4 expression in mononuclear cells were observed in peripheral blood during sepsis and it was found to be associated with high serum IL-6 levels and low MHC-Ⅱ expression in mononuclear cells,leading to increased mortality.
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 ofpGL3-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.