Figure 3

Activation of NF-κB after PM exposure. Exposure to PM from dairy farms stimulates NF-κB activity. A) Results from a gel shift assay illustrate the increased DNA-binding activity of NF-κB. U937 human macrophages were exposed for 1 h to 5 μg/ml and 10 μg/ml PM C (cutoff: 2.1 μm) collected on dairy farm #57. As a positive control, cells were exposed to pure endotoxin E.coli LPS (0.1 and 1 μg/ml LPS). The arrow indicates the formation of activated NF-κB complex binding to the NF-κB consensus DNA sequence. B) Activation of a NF-κB reporter plasmid after exposure to 10 μg/ml PM fraction C from dairy farm #57 in the absence or presence of a TLR4 neutralizing antibody (αTLR4) or 10 μg/ml Polymyxin B-treated PM C (PM C + PMB). In parallel, U937 macrophages were treated with particles from diesel engine exhaust (DEP) as a positive control for xenobiotic response element (XRE) reporter activity. Cells were transiently transfected with NF-κB or XRE luciferase reporter constructs and treated for 4 h with 10 μg/ml PM C or DEP.