RESUMO
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.
Assuntos
Antígenos de Superfície/imunologia , Inflamação/imunologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Antígenos CD/imunologia , Antígenos de Superfície/sangue , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Dexametasona/farmacologia , Feminino , Masculino , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
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.
Assuntos
Regulação para Baixo , Antígenos de Histocompatibilidade/metabolismo , Antígeno 96 de Linfócito/antagonistas & inibidores , Ativação de Macrófagos , Macrófagos/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/antagonistas & inibidores , Animais , Citocinas/agonistas , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Células HEK293 , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/genética , Humanos , Ligantes , Lipídeo A/toxicidade , Lipopolissacarídeos/toxicidade , Antígeno 96 de Linfócito/agonistas , Antígeno 96 de Linfócito/genética , Antígeno 96 de Linfócito/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Knockout , NF-kappa B/agonistas , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Células RAW 264.7 , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 3 Toll-Like/química , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
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.