The Amino-terminal Region of Toll-like Receptor 4 Is Essential for Binding to MD-2 and Receptor Translocation to the Cell Surface

Intense interest has centered around the role of a subset of regulatory T cells, CD4+CD25+ Treg, in controlling the development of auotimmune disorders, allograft rejection, infection, malignancy, and allergy. We previously reported that MD1, a molecule known to be important in regulation of expression of RP105, also was important in regulating alloimmunity, and that blockade of expression of MD1 diminished graft rejection in vivo. One mechanism by which an MD1-RP105 complex exerts an effect on immune responses is through interference with an LPS-derived signal delivered through the CD14-MD-2-TLR4 complex. We show below that LPS signaling for Treg induction occurs at higher LPS thresholds that for effector T cell responses. In addition, blockade of MD1 functional activity in dendritic cells (using anti-MD1 mAbs, MD1 antisense deoxyoligonucleotides, or responder cells from mice with deletion of the MD1 gene), resulted in elevated Treg induction in response to allogeneic stimulation (in vivo or in vitro) in the presence of LPS. These data offer one mechanistic explanation for the augmented immunosuppression described following anti-MD1 treatment.

Section: Cd25mentioning

confidence: 81%

MD1 Expression Regulates Development of Regulatory T Cells

Gorczynski

Yang

Miyake

2006

“…1B). In addition, both of these predicted proteins lack critical N-terminal amino acid residues that have been shown recently to be required for functional interaction with MD-2 and expression at the cell surface (40,41). It would be interesting to determine whether or not these isoforms function in cell types that purportedly respond to LPS via intracellular pathways (42,43).…”

Section: Discussionmentioning

confidence: 99%

MD-2 Mediates the Ability of Tetra-Acylated and Penta-Acylated Lipopolysaccharides to AntagonizeEscherichia coliLipopolysaccharide at the TLR4 Signaling Complex

Coats¹,

Pham²,

Bainbridge

et al. 2005

162

154

We have demonstrated previously that tetra-acylated LPS derived from the oral bacterium, Porphyromonas gingivalis, and penta-acylated msbB LPS derived from a mutant strain of Escherichia coli can antagonize the ability of canonical hexa-acylated E. coli LPS to signal through the TLR4 signaling complex in human endothelial cells. Activation of the TLR4 signaling complex requires the coordinated function of LPS binding protein (LBP), CD14, MD-2, and TLR4. To elucidate the specific molecular components that mediate antagonism, we developed a recombinant human TLR4 signaling complex that displayed efficient LPS-dependent antagonism of E. coli LPS in HEK293 cells. Notably, changes in the expression levels of TLR4 in HEK293 cells modulated the efficiency of antagonism by P. gingivalis LPS. Both soluble (s) CD14 and membrane (m) CD14 supported efficient P. gingivalis LPS-dependent and msbB LPS-dependent antagonism of E. coli LPS in the recombinant TLR4 system. When cells expressing TLR4, MD-2, and mCD14 were exposed to LPS in the absence of serum-derived LBP, efficient LPS-dependent antagonism of E. coli LPS was still observed indicating that LPS-dependent antagonism occurs downstream of LBP. Experiments using immunoprecipitates of sCD14 or sMD-2 that had been pre-exposed to agonist and antagonist indicated that LPS-dependent antagonism occurs partially at sCD14 and potently at sMD-2. This study provides novel evidence that expression levels of TLR4 can modulate the efficiency of LPS-dependent antagonism. However, MD-2 represents the principal molecular component that tetra-acylated P. gingivalis LPS and penta-acylated msbB LPS use to antagonize hexa-acylated E. coli LPS at the TLR4 signaling complex.

“…The molecular mechanisms by which TLR4 stays in the Golgi/ER and migrates to the cell surface are currently unknown. MD-2 has been shown not only to be essential for TLR4 binding with LPS but also indispensable for cell surface expression of TLR4 in many cell types, including HEK293 cells, bone marrow-derived dendritic cells, embryonic fibroblasts, and macrophages (17)(18)(19). Physical interaction between TLR4 and MD-2 was shown to be essential for the maturation of TLR4 and its presence in the cell membrane (17).…”

mentioning

confidence: 99%

Carbon Monoxide Suppresses Membrane Expression of TLR4 via Myeloid Differentiation Factor-2 in βTC3 Cells

Rocuts

Zhang

et al. 2010

Islet allografts from donor mice exposed to CO are protected from immune rejection after transplantation via the suppression of membrane trafficking/activation of TLR4 in islets/β cells. The molecular mechanisms of how CO suppresses TLR4 activation in β cells remain unclear and are the focus of this study. Cells of the insulinoma cell line, βTC3, were stably transfected with pcDNA3-TLR4-YFP and pDsRed-Monomer-Golgi plasmids and used to identify the subcellular distribution of TLR4 before and after LPS stimulation by confocal microscopy. Immunofluorescence analysis revealed that TLR4 mainly resides in the Golgi apparatus in βTC3 cells when in a quiescent state. LPS stimulation led to a rapid trafficking of TLR4 from the Golgi to the cell membrane. Physical interaction between TLR4 and myeloid differentiation factor-2 (MD-2) was confirmed by immunoprecipitation. Depleting MD-2 using small interfering RNA or blocking the N-glycosylation of cells using tunicamycin blocked membrane trafficking of TLR4. Pre-exposing cells to CO at a concentration of 250 parts per million suppressed membrane trafficking of TLR4 via inhibiting its glycosylation and the interaction between TLR4 and MD-2. In conclusion, MD-2 is required for the glycosylation of TLR4 and its consequent membrane trafficking in βTC3 cells. CO suppresses membrane activation of TLR4 via blocking its glycosylation and the physical interaction between TLR4 and MD-2.