High mobility group box 1 protein interacts with multiple Toll-like receptors

1 Division of Pulmonary Sciences and Critical Care Medicine and 2 Department of Surgery, University of Colorado Health Sciences Center, Denver, Colorado; and 3 Shino-Test Corporation, Kanagawa; 4 Laboratory and Vascular Medicine, Kagoshima University Graduate School of Medical Science and Dental Science, Kagoshima; and 5 Department of Medicine, Keio University, School of Medicine, Tokyo, Japan Submitted 9 August 2005 ; accepted in final form 26 October 2005 High mobility group box 1 (HMGB1), originally described as a DNA-binding protein, can also be released extracellularly and functions as a late mediator of inflammatory responses. Although recent reports have indicated that the receptor for advanced glycation end products (RAGE) as well as Toll-like receptor (TLR)2 and TLR4 are involved in cellular activation by HMGB1, there has been little evidence of direct association between HMGB1 and these receptors. To examine this issue, we used fluorescence resonance energy transfer (FRET) and immunoprecipitation to directly investigate cell surface interactions of HMGB1 with TLR2, TLR4, and RAGE. FRET images in RAW264.7 macrophages demonstrated association of HMGB1 with TLR2 and TLR4 but not RAGE. Transient transfections into human embryonic kidney-293 cells showed that HMGB1 induced cellular activation and NF- B-dependent transcription through TLR2 or TLR4 but not RAGE. Coimmunoprecipitation also found interaction between HMGB1 and TLR2 as well as TLR4, but not with RAGE. These studies provide the first direct evidence that HMGB1 can interact with both TLR2 and TLR4 and also supply an explanation for the ability of HMGB1 to induce cellular activation and generate inflammatory responses that are similar to those initiated by LPS. fluorescence resonance energy transfer; receptor of advanced glycation end products Address for reprint requests and other correspondence: E. Abraham, Div. of Pulmonary Sciences and Critical Care Medicine, Box C272, Univ. of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262 (e-mail: edward.abraham{at}uchsc.edu )