Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation

BACKGROUND: The transient receptor potential ankyrin 1 (TRPA1) channel, localized to airway sensory nerves, has been proposed to mediate airway inflammation evoked by allergen and cigarette smoke (CS) in rodents, via a neurogenic mechanism. However the limited clinical evidence for the role of neurogenic inflammation in asthma or chronic obstructive pulmonary disease raises an alternative possibility that airway inflammation is promoted by non-neuronal TRPA1. METHODOLOGY/PRINCIPAL FINDINGS: By using Real-Time PCR and calcium imaging, we found that cultured human airway cells, including fibroblasts, epithelial and smooth muscle cells express functional TRPA1 channels. By using immunohistochemistry, TRPA1 staining was observed in airway epithelial and smooth muscle cells in sections taken from human airways and lung, and from airways and lung of wild-type, but not TRPA1-deficient mice. In cultured human airway epithelial and smooth muscle cells and fibroblasts, acrolein and CS extract evoked IL-8 release, a response selectively reduced by TRPA1 antagonists. Capsaicin, agonist of the transient receptor potential vanilloid 1 (TRPV1), a channel co-expressed with TRPA1 by airway sensory nerves, and acrolein or CS (TRPA1 agonists), or the neuropeptide substance P (SP), which is released from sensory nerve terminals by capsaicin, acrolein or CS), produced neurogenic inflammation in mouse airways. However, only acrolein and CS, but not capsaicin or SP, released the keratinocyte chemoattractant (CXCL-1/KC, IL-8 analogue) in bronchoalveolar lavage (BAL) fluid of wild-type mice. This effect of TRPA1 agonists was attenuated by TRPA1 antagonism or in TRPA1-deficient mice, but not by pharmacological ablation of sensory nerves. CONCLUSIONS: Our results demonstrate that, although either TRPV1 or TRPA1 activation causes airway neurogenic inflammation, solely TRPA1 activation orchestrates an additional inflammatory response which is not neurogenic. This finding suggests that non-neuronal TRPA1 in the airways is functional and potentially capable of contributing to inflammatory airway diseases

Potency (EC₅₀ and CI) of the various TRPA1 agonists in different non-neuronal cell types of the human respiratory tract.

<p>Potency (EC₅₀ and CI) of the various TRPA1 agonists in different non-neuronal cell types of the human respiratory tract.</p

TRPA1 mediates IL-8 release by acrolein from human airway/lung cells in primary culture.

<p>Overnight exposure to acrolein (ACR) induces IL-8 release from small airway epithelial cells (SAEC) (<b>A</b>), normal human lung fibroblasts (NHLF) (<b>B</b>) and human bronchial smooth muscle cells (HBSMC) (<b>C</b>) in a concentration–dependent manner. IL-8 release evoked by ACR is reduced by HC-030031 (HC, 30 µM) and AP18 (10 µM). Each column represents mean ± SEM of at least 3 independent experiments. ^§<i>P</i><0.05 vs. Basal group or Veh/Veh-ACR; ^*<i>P</i><0.05 vs. Veh/ACR.</p

Functional TRPA1 is expressed in human airway/lung cells.

<p>Intracellular calcium response was used to assess agonist-induced TRPA1 activation in small airways epithelial cells (SAEC) (<b>A</b>), normal human lung fibroblasts (NHLF) (<b>B</b>) and bronchial smooth muscle cells (HBSMC) (<b>C</b>). Typical traces and pooled data of the concentration-dependent calcium response evoked by the selective TRPA1 agonists, cinnamaldehyde (CNM, typical traces and black circles) and acrolein (ACR, grey circles), in all different cell types in primary culture. Similarly to CNM and ACR, cigarette smoke extract (CSE, black triangles) produces in all the different types of cells a concentration-dependent calcium response. Responses to CNM, ACR and CSE are prevented by selective TRPA1 antagonists, HC-030031 (HC, 10 µM) and AP18 (10 µM). The activating peptide (SLIGKV-NH₂) of the PAR-2 receptor (PAR-2 AP, 100 µM) elicits a calcium response that is not modified by TRPA1 antagonists. Veh is a combination of vehicles of HC and AP18. Values represent mean ± SEM of n>25 cells. ^*<i>P</i><0.05 <i>vs.</i> Veh.</p

TRPA1 channel expression in non-neuronal cells of the human and mouse respiratory tract.

<p>(<b>A</b>) Total RNAs were extracted from primary small airways epithelial cells (SAEC), human type II alveolar epithelial cells (A549), human primary smooth muscle cells (HBSMC), human embryonic lung fibroblasts (IMR90) and primary normal human lung fibroblasts (NHLF) and relative TRPA1 mRNA amounts were measured by Taqman Real-Time PCR assay. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042454#s2" target="_blank">Results</a> are normalized to the reference gene, β-actin. Each column represents mean ± SEM of n>2 independent experiments. Immunohistochemical analysis of TRPA1 expression in samples taken from human (<b>B</b>) or <i>Trpa1^+/+</i> and <i>Trpa1^−/−</i> mouse airways and lung (<b>C</b>). Representative images of TRPA1 immunostaining show intense staining in epithelial and smooth muscle cells in human tissue. No staining is detected in human samples incubated with the normal serum peptide (Negative control). (<b>C</b>) Incubation with TRPA1 antibody shows a strong staining in epithelial and smooth muscle cells in tissues taken from <i>Trpa1^+/+</i> mice, but not in those from <i>Trpa1^−/−</i> mice. Preadsorption of the TRPA1 antibody with the peptide used for immunization abolished staining (Peptide). Staining for cytokeratin and α-smooth muscle actin (α-SMA) overlaps with the TRPA1 staining in the bronchial epithelium and smooth muscle layer in serial section of human and mice airways/lung tissues (<b>B</b> and <b>C</b>). Scale bar 100 µm.</p