TLR4 and MD-2 Expression Is Regulated by Immune-mediated Signals in Human Intestinal Epithelial Cells*

Abstract

The normal intestinal epithelium is not inflamed despite contact with a high density of commensal bacteria. Intestinal epithelial cells (IEC) express low levels of TLR4 and MD-2 and are lipopolysaccharide (LPS)-unresponsive. We hypothesized that immune-mediated signals regulate the expression of TLR4 and MD-2 in IEC. Expression of TLR4 and MD-2 was examined in normal colonic epithelial cells or intestinal epithelial cell lines. The effect of the cytokines interferon (IFN)-γ, IFN-α, and tumor necrosis factor-α (TNF-α) on TLR4 and MD-2 expression was examined by reverse transcription-PCR and Western blot. NF-κB transcriptional activation and interleukin-8 secretion were used as measures of LPS responsiveness. Native colonic epithelial cells and IEC lines express a low level of TLR4 and MD-2 mRNA. IFN-γ regulates MD-2 expression in both IEC lines, whereas IFN-γ and TNF-α regulate TLR4 mRNA expression in IEC lines. Pre-incubation with IFN-γ and/or TNF-α sensitizes IEC to LPS-dependent interleukin-8 secretion. To examine MD-2 transcriptional regulation, we cloned a 1-kb sequence proximal to the MD-2 gene translational start site. This promoter directed expression of a reporter gene in endothelial cells and IEC. IFN-γ positively regulated MD-2 promoter activity in IEC. Co-expression of a STAT inhibitor, SOCS3, blocked IFN-γ-mediated MD-2 promoter activation. T cell-derived cytokines lead to increased expression of TLR4 and MD-2 and LPS-dependent pro-inflammatory cytokine secretion in IEC. IFN-γ regulates expression of the critical TLR4 co-receptor MD-2 through the Janus tyrosine kinase-STAT pathway. Th1 cytokines may initiate or perpetuate intestinal inflammation by altering toll-like receptor expression and bacterial reactivity.

  • Abbreviations:
    LPS
    lipopolysaccharide
    TLR
    toll-like receptor
    IEC
    intestinal epithelial cell(s)
    IFN
    interferon
    JAK
    Janus tyrosine kinase
    STAT
    signal transducers and activators of transcription
    IRF
    interferon response factor
    contig
    group of overlapping clones
    IL
    interleukin
    ELISA
    enzyme-linked immunosorbent assay
    GAPDH
    glyceraldehyde-3-phosphate dehydrogenase
    FBS
    fetal bovine serum
    Pen/Strep
    penicillin/streptomycin
    RT
    reverse transcription
    GAS
    interferon-γ activation site
    HMEC
    human dermal endothelial cell
    • Received October 26, 2001.
    • Revision received March 12, 2002.
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    This Article

    1. The Journal of Biological Chemistry 277, 20431-20437.
    1. All Versions of this Article:
      1. M110333200v1
      2. 277/23/20431 (most recent)

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