Mechanisms of asthma and allergic inflammation
Toll-like receptor 2, 3, and 4 expression and function in human airway smooth muscle

https://doi.org/10.1016/j.jaci.2006.05.013Get rights and content

Background

Host defense against microbial pathogens is elicited through the innate immune system by means of Toll-like receptors (TLRs). Airway smooth muscle cells (ASMCs) display proinflammatory and immunomodulatory functions. ASMCs might participate in airway inflammatory responses associated with innate immune activation.

Objectives

We determined the effects of cytokines, TLR ligands, and corticosteroids on TLR expression and function in human ASMCs.

Methods

Real-time PCR and flow cytometry were used to assess TLR mRNA and protein expression, respectively. ASMCs were stimulated with TLR ligands, and chemokine release was measured by means of ELISA.

Results

ASMCs expressed TLR1 to TLR10 mRNA, and TLR2 and TLR3 protein expression was demonstrated. TNF-α and double-stranded RNA (dsRNA; TLR3 ligand) were potent inducers of TLR2 and TLR3 mRNA expression, and both stimuli had additive or synergistic effects with IFN-γ on TLR2 and TLR4, but not TLR3, mRNA expression. Peptidoglycan (TLR2 ligand) and LPS (TLR4 ligand) weakly enhanced TLR2 mRNA expression. Peptidoglycan, dsRNA, and LPS induced IL-8 and eotaxin release, with dsRNA being most potent. dsRNA also modulated cytokine-induced chemokine release in a differential manner. Dexamethasone inhibited cytokine- and ligand-induced TLR2, TLR3, and TLR4 expression and chemokine release. However, dexamethasone potentiated TLR2 expression induced by combined IFN-γ and TNF-α stimulation.

Conclusion

Expression of TLR2, TLR3, and TLR4 is regulated by cytokines and TLR ligands, and their activation mediates chemokine release in ASMCs.

Clinical implications

Proinflammatory responses mediated by activation of pathogen-recognition receptors in ASMCs might contribute to infectious exacerbations of airway inflammatory conditions, such as asthma and chronic obstructive pulmonary disease.

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.

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