Co2+ mediates its proinflammatory effects via TLR4

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Metal allergens nickel and cobalt facilitate TLR4 homodimerization independently of MD2 Co2+ mediates its proinflammatory effects via TLR4. (A–C) Keratinocytes lack functional TLR4 expression. (A) Enzyme‐linked immunosorbent assay showing IL‐8 production in NHEK exposed for 8 h to LPS (S. minnesota R595; 1 μg ml−1), IL‐1β (100 U ml−1), TNF (10 ng ml−1) or medium (Ctrl). (B) Quantitative real‐time PCR analysis of human MD2 (black bars) and TLR4 (grey bars) expression in unstimulated NHEK as compared with TLR4/MD2‐negative HEK293 cells or TLR4/MD2‐positive HUVEC. Data are presented as average‐fold mRNA expression±s.d. related to the HUVEC‐positive control (set to 100%). (C) Enzyme‐linked immunosorbent assay, showing IL‐8 protein release from NHEK transfected with human MD2, TLR4 or both in combination. Cells were stimulated for 8 h with 1.5 mM Co2+, 1.5 mM Ni2+, 1 μg ml−1 LPS or medium (Ctrl). (D–F) Co2+ requires TLR4/MD2 for proinflammatory signalling. Cells were stimulated as in (C). (D) IL‐8 production by HUVEC transfected with the indicated small interfering RNAs for 48 h before stimulation. (E) Co2+‐induced IL‐8 protein expression in the indicated cell lines as determined by western blot analysis of total lysates. (F) Co2+‐induced activity of a transfected 6 × κB‐luciferase (luc) reporter on transfection of HEK293 MD2 cells with TLR4, TLR4 H456A/H458A, Tlr4 or a ‘humanized’ Tlr4 mutant with histidine substitutions at the corresponding positions of the mouse sequence (Tlr4 Y454H/N456H). Bar diagrams represent averages (A–D) or mean fold values (F) of three to four independent experiments±s.d. The immunoblot in (E) is representative of three independent experiments. P>0.05 not significant (NS), *P<0.05, **P<0.01, ***P<0.001, t‐test. HUVEC, human primary umbilical vein endothelial cells; IL, interleukin; LPS, lipopolysaccharide; NHEKs, normal human epidermal keratinocytes; NS, not significant; TLR4, Toll‐like receptor 4; TNF, tumour‐necrosis factor. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO Rep, Volume: 13, Issue: 12, Pages: 1109-1115, First published: 12 October 2012, DOI: (10.1038/embor.2012.155)