Signaling of High Mobility Group Box 1 (HMGB1) through Toll-like Receptor 4 in Macrophages Requires CD14

Sodam Kim,Kevin P Mollen,Melanie J Scott,Patricia Loughran,Richard Shapiro,Sun Young Kim,Timothy R Billiar,John P Pribis,Michael Lotze

doi:10.2119/molmed.2012.00306

Sodam Kim, Kevin P Mollen + Show 7 more

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https://doi.org/10.2119/molmed.2012.00306

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Abstract

High mobility group box 1 (HMGB1) is a DNA-binding protein that possesses cytokinelike, proinflammatory properties when released extracellularly in the C23-C45 disulfide form. HMGB1 also plays a key role as a mediator of acute and chronic inflammation in models of sterile injury. Although HMGB1 interacts with multiple pattern recognition receptors (PRRs), many of its effects in injury models occur through an interaction with toll-like receptor 4 (TLR4). HMGB1 interacts directly with the TLR4/myeloid differentiation protein 2 (MD2) complex, although the nature of this interaction remains unclear. We demonstrate that optimal HMGB1-dependent TLR4 activation in vitro requires the coreceptor CD14. TLR4 and MD2 are recruited into CD14-containing lipid rafts of RAW264.7 macrophages after stimulation with HMGB1, and TLR4 interacts closely with the lipid raft protein GM1. Furthermore, we show that HMGB1 stimulates tumor necrosis factor (TNF)-α release in WT but not in TLR4(-/-), CD14(-/-), TIR domain-containing adapter-inducing interferon-β (TRIF)(-/-) or myeloid differentiation primary response protein 88 (MyD88)(-/-) macrophages. HMGB1 induces the release of monocyte chemotactic protein 1 (MCP-1), interferon gamma-induced protein 10 (IP-10) and macrophage inflammatory protein 1α (MIP-1α) in a TLR4- and CD14-dependent manner. Thus, efficient recognition of HMGB1 by the TLR4/MD2 complex requires CD14.

Highlights

The toll-like receptors (TLRs) are a major family of pattern recognition receptors (PRRs) that reside in cell membranes, both at the cell surface and in endosomes, that recognize and respond to a variety of bacterial products, called pathogen-associated molecular patterns (PAMPs) [1]
RAW264.7 cells were stimulated for 18 h with phosphatebuffered saline (PBS) or 0.1, 0.5 or 1 ng/mL LPS; and tumor necrosis factor (TNF)-α levels in supernatants were measured by enzyme-linked immunosorbent assay (ELISA)
TNF-α induced by 1 ng/mL LPS was completely abrogated by concurrent treatment with 10 μg/mL polymyxin B (PMB)

Summary

Introduction

The toll-like receptors (TLRs) are a major family of pattern recognition receptors (PRRs) that reside in cell membranes, both at the cell surface and in endosomes, that recognize and respond to a variety of bacterial products, called pathogen-associated molecular patterns (PAMPs) [1]. Recent studies show that only HMGB1 in which cysteine 106 is maintained in the thiol state, and in whcih cysteines 23 and 45 form a disulfide bond, is capable of activating TLR4 signaling [13,14,15]. It is unknown whether HMGB1 recognition by the TLR4/ myeloid differentiation protein 2 (MD2) complex shares similarities with other prototypical activators of TLR4 signaling

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