Signal transducer and activator of transcription 6 down-regulates toll-like receptor-4 expression of a monocytic cell line

• Published in: Clinical and experimental allergy Vol. 36; no. 2; pp. 158 - 165
• Main Authors: Fiset, P. O., Tulic, M. K., Skrablin, P. S. A., Grover, S. M., Létuvé, S., Mazer, B. D., Hamid, Q.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-02-2006; Blackwell; Wiley Subscription Services, Inc
• Subjects: Adult; Allergic diseases; atopy; Biological and medical sciences; Cell Line; Down-Regulation; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; IL-4; Immunopathology; Interleukin-4 - pharmacology; Lipopolysaccharides; Medical sciences; monocytes; Monocytes - metabolism; NF-kappa B - metabolism; Protein-Tyrosine Kinases - antagonists & inhibitors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - analysis; STAT6; STAT6 Transcription Factor - metabolism; TLR4; Toll-Like Receptor 4 - analysis; Toll-Like Receptor 4 - genetics; Toll-Like Receptor 4 - metabolism; Transfection - methods; Tyrphostins - pharmacology; Immunopathology; Allergy; monocytes; Toll like receptor 4; Monocyte; Cytokine; IL-4; Atopy; Interleukin 4; Immunology; Transcription factor STAT6; Established cell line; STAT6; TLR4
• DOI: 10.1111/j.1365-2222.2006.02370.x
• EISSN: 1365-2222
• ISSN: 0954-7894

Summary
Background
Toll‐like receptor 4 (TLR4), part of the bacterial lipopolysaccharide (LPS) receptor, is an important bridge between innate and adaptive immunity. Our previous studies have indicated reduced expression of TLR4 and reduced responsiveness to LPS in nasal mucosa of atopic adults compared with non‐atopic adults. IL‐4 and signal transducer and activator of transcription 6 (STAT6), which are increased in atopic patients, may have a role in modulating TLR4.
Objective
To examine direct effects of IL‐4 and STAT6 on TLR4 expression of U‐937 monocytic cells.
Methods
LPS responsiveness, under different conditions of U‐937 cells was measured by nuclear factor (NF)‐κB activation of transcription. TLR4 mRNA was quantified by real‐time PCR and TLR4 surface expression was measured by flow cytometry. The promoter and 4.3 kb of the upstream region of TLR4 were cloned into a plasmid vector and transiently transfected into U‐937 cells. Transfected cells were incubated with IL‐4 and transcriptional activity was assayed by the luciferase assay. STAT6 was transfected to evaluate overexpression of this transcription factor. Cells were also incubated with Tyrphostin AG490 to inhibit tyrosine kinases.
Results
NF‐κB activation by LPS was inhibited by IL‐4 pre‐incubation but not when IL‐4 was added at the same time as LPS. TLR4 mRNA expression was inhibited by IL‐4 as early as 6 h but the effect was lost by 24 h. Surface expression of TLR4 was inhibited by IL‐4 at 12 and 24 h, but returned to baseline at 48 h. IL‐4 inhibited activity of the TLR4 promoter as early as 6 h, but, like the mRNA, these effects were transient. STAT6 overexpression enhanced the inhibition of the TLR4 promoter and prolonged it. Inhibition of TLR4 by IL‐4 was abolished by pre‐incubation with the tyrosine kinase inhibitor Tyrphostin AG490.
Conclusion
Our findings demonstrate that IL‐4, through STAT6, can modulate TLR4 expression and suggests that Th2 cytokines can impact on the LPS responsiveness of cells.