ABSTRACT
Recently, we reported a novel function for C4b-binding protein (C4BP) in inhibiting the toll-like receptor (TLR)1/2 response by interacting with TLR2. TLRs share a common structure; hence, we examined the effect of C4BP on activation of other TLRs-TLR4 and TLR3. The results of immunoprecipitation assays suggest that C4BP interacts with TLR4/MD-2 but not TLR3. C4BP inhibits TLR4/MD-2-mediated, but not TLR3-mediated, proinflammatory cytokine production and nuclear factor (NF)-κB signaling. C4BP-deficient mice show increased interleukin (IL)-6 production in response to the TLR4/MD-2 ligand. A competition assay revealed that C4BP prevents an interaction between TLR4/MD-2 and its ligand. These findings indicate that C4BP binds to cell surface TLRs and inhibits the TLR-TLR ligand interaction, thereby inhibiting TLR activation.
Subject(s)
Down-Regulation , Histocompatibility Antigens/metabolism , Lymphocyte Antigen 96/antagonists & inhibitors , Macrophage Activation , Macrophages/metabolism , Signal Transduction , Toll-Like Receptor 4/antagonists & inhibitors , Animals , Cytokines/agonists , Cytokines/antagonists & inhibitors , Cytokines/metabolism , Down-Regulation/drug effects , Female , HEK293 Cells , Histocompatibility Antigens/chemistry , Histocompatibility Antigens/genetics , Humans , Ligands , Lipid A/toxicity , Lipopolysaccharides/toxicity , Lymphocyte Antigen 96/agonists , Lymphocyte Antigen 96/genetics , Lymphocyte Antigen 96/metabolism , Macrophage Activation/drug effects , Macrophages/drug effects , Macrophages/immunology , Mice , Mice, Knockout , NF-kappa B/agonists , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , RAW 264.7 Cells , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Signal Transduction/drug effects , Toll-Like Receptor 3/chemistry , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 4/agonists , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolismABSTRACT
TLR2 associates with TLR1 and recognizes microbial lipoproteins. Pam3CSK4, a triacylated lipoprotein, is anchored to the extracellular domain of TLR1 and TLR2 and induces pro-inflammatory signals. Here we show that C4b binding protein (C4BP), which is a complement pathway inhibitor, is a TLR2-associated molecule. Immunoprecipitation assay using anti-TLR2 mAb shows that C4BP binds to TLR2. In C4BP-deficient mice, Pam3CSK4-induced IL-6 levels were increased compared with wild type mice. In C4BP-expressing cells, Pam3CSK4-induced IL-8 production was reduced depending on the C4BP expression levels. These results reveal the important role of C4BP in negative regulation of TLR1/2-dependent pro-inflammatory cytokine production. Furthermore, using a fluorescent conjugated Pam3CSK4, we show that C4BP blocks the binding of Pam3CSK4 to TLR1/2. Finally, we show that exogenous C4BP also inhibits Pam3CSK4-induced signaling leading to IL-8 production. Our results indicate C4BP binding to TLR2 and consequent neutralization of its activity otherwise inducing pro-inflammatory cytokine production. C4BP is a negative regulator of TLR1/2 activity.
Subject(s)
Complement C4b-Binding Protein/metabolism , Toll-Like Receptor 1/metabolism , Toll-Like Receptor 2/metabolism , Animals , Binding Sites , Complement Activation , HEK293 Cells , Humans , Interleukin-6/metabolism , Interleukin-8/metabolism , Lipopeptides/metabolism , Mice , Mice, Inbred C57BL , Protein Binding , Signal TransductionABSTRACT
Radioprotective 105 (RP105) is a type I transmembrane protein, which associates with a glycoprotein, MD-1. Monoclonal antibody (mAb)-mediated ligation of RP105/MD-1 robustly activates B cells. RP105/MD-1 is structurally similar to Toll-like receptor 4 (TLR4)/MD-2. B-cell responses to TLR2 and TLR4/MD-2 ligands are impaired in the absence of RP105 or MD-1. In addition to RP105/MD-1, MD-1 alone is secreted. The structure of MD-1 shows that MD-1 has a hydrophobic cavity that directly binds to phospholipids. Little is known, however, about a ligand for MD-1 and the role of MD-1 in vivo To study the role of RP105/MD-1 and MD-1 alone, specific mAbs against MD-1 are needed. Here, we report the establishment and characterization of two anti-MD-1 mAbs (JR2G9, JR7G1). JR2G9 detects soluble MD-1, whereas JR7G1 binds both soluble MD-1 and the cell surface RP105/MD-1 complex. With these mAbs, soluble MD-1 was detected in the serum and urine. The MD-1 concentration was altered by infection, diet and reperfusion injury. Serum MD-1 was rapidly elevated by TLR ligand injection in mice. The quantitative PCR and supernatant-precipitated data indicate that macrophages are one of the sources of serum soluble MD-1. These results suggest that soluble MD-1 is a valuable biomarker for inflammatory diseases.
