HMGB1 signals through toll-like receptor (TLR) 4 and TLR2.
In response to bacterial endotoxin (e.g., LPS) or endogenous proinflammatory cytokines (e.g., TNF and IL-1beta), innate immune cells release HMGB1, a late cytokine mediator of lethal endotoxemia and sepsis. The delayed kinetics of HMGB1 release makes it an attractive therapeutic target with a wider window of opportunity for the treatment of lethal systemic inflammation. However, the receptor(s) responsible for HMGB1-mediated production of proinflammatory cytokines has not been well characterized. Here we demonstrate that in human whole blood, neutralizing antibodies against Toll-like receptor 4 (TLR4, but not TLR2 or receptor for advanced glycation end product) dose-dependently attenuate HMGB1-induced IL-8 release. Similarly, in primary human macrophages, HMGB1-induced TNF release is dose-dependently inhibited by anti-TLR4 antibodies. In primary macrophages from knockout mice, HMGB1 activates significantly less TNF release in cells obtained from MyD88 and TLR4 knockout mice as compared with cells from TLR2 knockout and wild-type controls. However, in human embryonic kidney 293 cells transfected with TLR2 or TLR4, HMGB1 effectively induces IL-8 release only from TLR2 overexpressing cells. Consistently, anti-TLR2 antibodies dose-dependently attenuate HMGB1-induced IL-8 release in human embryonic kidney/TLR2-expressing cells and markedly reduce HMGB1 cell surface binding on murine macrophage-like RAW 264.7 cells. Taken together, our data suggest that there is a differential usage of TLR2 and TLR4 in HMGB1 signaling in primary cells and in established cell lines, adding complexity to studies of HMGB1 signaling which was not previously expected.
Yu
Man
M
Laboratories of Biomedical Science, The Feinstein Institute for Medical Research, Manhasset, NY 11030, USA.
Wang
Haichao
H
Ding
Aihao
A
Golenbock
Douglas T
DT
Latz
Eicke
E
Czura
Christopher J
CJ
Fenton
Matthew J
MJ
Tracey
Kevin J
KJ
Yang
Huan
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001773:Blood Cells / Q000378:metabolism; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004306:Dose-Response Relationship, Immunologic; D017127:Glycation End Products, Advanced; D024243:HMGB1 Protein / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017127:Glycation End Products, Advanced; D024243:HMGB1 Protein; D016209:Interleukin-8; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha