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

Hiroki Tsukamoto, Kenji Fukudome, Shoko Takao, Naoko Tsuneyoshi, Hideyuki Ihara, Yoshitaka Ikeda, Masao Kimoto

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)495-506
Number of pages12
JournalInternational immunology
Volume24
Issue number8
DOIs
Publication statusPublished - 2012 Aug

Keywords

  • Inhibitory mAb
  • Innate immunity
  • MD-2
  • Toll-like receptor 4

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Fingerprint

Dive into the research topics of 'Multiple potential regulatory sites of TLR4 activation induced by LPS as revealed by novel inhibitory human TLR4 mAbs'. Together they form a unique fingerprint.

Cite this