Multiple potential regulatory sites of TLR4 activation induced by LPS as revealed by novel inhibitory human TLR4 mAbs

• Published in: International immunology Vol. 24; no. 8; pp. 495 - 506
• Main Authors: Tsukamoto, Hiroki, Fukudome, Kenji, Takao, Shoko, Tsuneyoshi, Naoko, Ihara, Hideyuki, Ikeda, Yoshitaka, Kimoto, Masao
• Format: Journal Article
• Language: English
• Published: England 01-08-2012
• Subjects: Animals; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - pharmacology; Autoimmunity; Blotting, Western; CD86 antigen; Cell Line; Cytokines; Deletion mutant; Drugs; Enzyme Activation - drug effects; Enzyme Activation - immunology; Epitopes; Fc receptors; Flow Cytometry; Fluorescent Antibody Technique; Humans; Immune response; Immunity, Innate - immunology; Immunoprecipitation; Lipopolysaccharides; Lipopolysaccharides - immunology; Lymphocyte Antigen 96 - drug effects; Lymphocyte Antigen 96 - immunology; Lymphocyte Antigen 96 - metabolism; Mice; Monoclonal antibodies; Reverse Transcriptase Polymerase Chain Reaction; Septic shock; Signal Transduction - drug effects; Signal Transduction - immunology; TLR4 protein; Toll-Like Receptor 4 - drug effects; Toll-Like Receptor 4 - immunology; Toll-Like Receptor 4 - metabolism; Toll-like receptors; Transfection
• ISSN: 0953-8178; 1460-2377
• DOI: 10.1093/intimm/dxs053

Recognition of LPS by the toll-like receptor 4 (TLR4)/MD-2 complex is a trigger of innate immune defense against bacterial invasion. However, excessive immune activation by this receptor complex causes septic shock and autoimmunity. Manipulation of TLR4 signaling represents a potential therapy that would avoid the detrimental consequences of unnecessary immune responses. In this study, we established two novel mAbs that inhibit LPS-induced human TLR4 activation. HT52 and HT4 mAbs inhibited LPS-induced nuclear factor-κB activation in TLR4/MD-2-expressing Ba/F3-transfected cells and cytokine production and up-regulation of CD86 in the human cell line U373 and PBMCs. These inhibitory activities were stronger than that of HTA125 mAb, which we previously reported. Immunofluorescent and biochemical studies using TLR4 deletion mutants revealed that HT52 and HT4 recognized spatially distinct regions on TLR4 irrespective of MD-2 association. The HT52 and HTA125 epitopes were localized within aa 50-190, while the HT4 epitope was formed only by the full length of TLR4. In addition, we demonstrated that HT52 and HT4 failed to compete with LPS for binding to TLR4/MD-2 but inhibited LPS-induced TLR4 internalization. Inhibitory activities were not due to the interaction with the Fcγ receptor CD32. Our finding that binding of mAbs to at least two distinct regions on TLR4 inhibits LPS-dependent activation provides a novel method for manipulating TLR4 activation and also a rationale for designing drugs targeted to TLR4.