Mechanisms of asthma and allergic inflammationToll-like receptor 2, 3, and 4 expression and function in human airway smooth muscle
Section snippets
Materials
Recombinant human cytokines were purchased from R&D systems (Abingdon, United Kingdom). A synthetic oligodeoxynucleotide that contains CpG motifs mimicking bacterial DNA (5′-ggGGGACGATCGTCgggggG-3′; the small letters indicate phosphorothioate linkages, the capital letters indicate phosphodiester linkages 3′ of the base, and the bold letters indicate CpG dinucleotides) and primers for TLR1 through TLR10,10 glyceraldehyde-3-phosphate dehydrogenase, and 18S were purchased from Sigma Genosys
TLR1 through TLR10 mRNA expression under basal conditions and after stimulation with cytokines and TLR ligands
Using quantitative RT-PCR, we show that ASMCs express TLR1 through TLR10 mRNA under basal conditions. Threshold cycle numbers for TLR2, TLR3, and TLR6 were less than 25, whereas those for TLR1, TLR5, TLR7, TLR9, and TLR10 were equal to or less than 30. TLR4 and TLR8 both had threshold cycle numbers of less than 35 (Fig 1, A). Stimulation of ASMCs with TNF-α (10 ng/mL) for 24 hours increased TLR1 through TLR7 and TLR10 mRNA expression between 2- and 30-fold. IL-1β (10 ng/mL) was a very weak
Discussion
We have shown that human ASMCs express TLR1 through TLR10 mRNA in the basal state and that, with the exception of TLR8 and TLR9, their expression is increased by TNF-α. In more detailed studies we demonstrate that IFN-γ, TNF-α, and the TLR3 ligand dsRNA have significant regulatory effects on TLR2, TLR3, and TLR4 expression. Stimulation of ASMCs with TLR2, TLR3, or TLR4 ligands leads to IL-8 and eotaxin release, and dsRNA differentially regulates chemokine release, causing inhibition or
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Supported by a grant from the Wellcome Trust.
Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.