Epinephrine Activation of the β₂-Adrenoceptor Is Required for IL-13-Induced Mucin Production in Human Bronchial Epithelial Cells

<div><p>Mucus hypersecretion by airway epithelium is a hallmark of inflammation in allergic asthma and results in airway narrowing and obstruction. Others have shown that administration a T_H2 cytokine, IL-13 is sufficient to cause mucus hypersecretion <i>in vivo</i> and <i>in vitro</i>. Asthma therapy often utilizes β₂-adrenoceptor (β₂AR) agonists, which are effective acutely as bronchodilators, however chronic use may lead to a worsening of asthma symptoms. In this study, we asked whether β₂AR signaling in normal human airway epithelial (NHBE) cells affected mucin production in response to IL-13. This cytokine markedly increased mucin production, but only in the presence of epinephrine. Mucin production was blocked by ICI-118,551, a preferential β₂AR antagonist, but not by CGP-20712A, a preferential β₁AR antagonist. Constitutive β₂AR activity was not sufficient for IL-13 induced mucin production and β-agonist-induced signaling is required. A clinically important long-acting β-agonist, formoterol, was as effective as epinephrine in potentiating IL-13 induced MUC5AC transcription. IL-13 induced mucin production in the presence of epinephrine was significantly reduced by treatment with selective inhibitors of ERK1/2 (FR180204), p38 (SB203580) and JNK (SP600125). Replacement of epinephrine with forskolin + IBMX resulted in a marked increase in mucin production in NHBE cells in response to IL-13, and treatment with the inhibitory cAMP analogue Rp-cAMPS decreased mucin levels induced by epinephrine + IL-13. Our findings suggest that β₂AR signaling is required for mucin production in response to IL-13, and that mitogen activated protein kinases and cAMP are necessary for this effect. These data lend support to the notion that β₂AR-agonists may contribute to asthma exacerbations by increasing mucin production via activation of β₂ARs on epithelial cells.</p></div

Agonist induced β₂AR signaling is required for mucin production in response to IL-13 in NHBE cells.

<p>NHBE cells were grown in the presence of 3 μM epinephrine, then at ALI, they were treated with 20 ng/ml IL-13 in combination with 10 μM nadolol (a non-selective inverse agonist of βARs) or 10 μM alprenolol (a non-selective β₂AR blocker with no inverse agonist activity) for 14 days. <b>A</b>: MUC5AC transcripts were measured by qRT-PCR. Data are presented as fold change compared to cells grown in the presence of epinephrine only. <b>B</b>: Quantification of intracellular mucin 5AC content. The ratio of mucin 5AC integrated density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132559#pone.0132559.s004" target="_blank">S4A Fig</a>). <b>C</b>: Quantification of intracellular mucin glycoproteins in response to different ligands. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to control cells. See the supplement for the representative images (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132559#pone.0132559.s004" target="_blank">S4B Fig</a>). Data are presented as means ± SEM from three donors. * and # indicate p<0.05 significance as compared to + epinephrine and + epinephrine + IL-13 treated cells respectively.</p

Epinephrine is required for mucin production in response to IL-13 in NHBE cells.

<p><b>A</b>: NHBE cells were grown in the presence or absence of 3 μM epinephrine. At ALI, the cells were treated with 20 ng/ml IL-13 for 14 days, total RNA was harvested and then MUC5AC transcripts were measured by qRT-PCR. Data are presented as fold change compared to the corresponding treatment control (in the absence of IL13). <b>B</b>: Representative images of immunofluorescence with a rabbit antibody against human mucin 5AC (red) (scale bar = 100 μm). The Transwell membranes were incubated with DAPI to counterstain the nuclei (blue). Incubation with antibody diluent showed no red fluorescence (data not shown). The ratio of integrated fluorescence density of each group to the integrated mucin 5AC density of the corresponding control group was calculated and expressed as fold change. <b>C</b>: PAFS staining of NHBE cells to quantify total intracellular mucin glycoproteins. Representative images are shown. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to the corresponding control cells (in the absence of IL-13 treatment). Data are presented as means ± SEM from three donors. *, † and ¥ indicate p<0.05 significance as compared to + epinephrine,−epinephrine and −epinephrine + IL-13 treated cells respectively.</p

Inhibiting PKA signaling reduced mucin production in response to IL-13 in NHBE cells.

<p>Cells were grown in the presence of 3 μM epinephrine, then after ALI, they were treated with 20 ng/ml IL-13 in combination with 50 μM or 100 μM Rp-cAMP (cAMP-dependent protein kinase inhibitor) for 14 days. <b>A</b>: MUC5AC transcripts were measured by qRT-PCR, and the data presented as fold change compared to cells grown in the absence of inhibitor. <b>B</b>: Quantification of intracellular mucin 5AC content. The ratio of mucin 5AC integrated density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132559#pone.0132559.s006" target="_blank">S6A Fig</a>). <b>C</b>: Quantification of intracellular mucin glycoproteins. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to control cells. See the supplement for the representative images (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132559#pone.0132559.s006" target="_blank">S6B Fig</a>). Data are presented as means ± SEM from three donors. * and # indicate p<0.05 significance as compared to + epinephrine and + epinephrine + IL-13 treated cells respectively.</p

cAMP potentiates mucin production in response to IL-13 in NHBE cells.

<p>Cells were grown in the absence of epinephrine, then at ALI, they were incubated with or without 20 ng/ml IL-13 with or without 10 μM forskolin and 100 μM IBMX for 14 days. <b>A</b>: MUC5AC transcripts were measured by qRT-PCR, and the data presented as fold change compared to cells grown in the absence of IL-13, IBMX and forskolin (control cells) <b>B</b>: Quantification of intracellular mucin 5AC content. The ratio of mucin 5AC integrated density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132559#pone.0132559.s007" target="_blank">S7A Fig</a>). <b>C</b>: Quantification of intracellular mucin glycoproteins. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to control cells. See the supplement for the representative images (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132559#pone.0132559.s007" target="_blank">S7B Fig</a>). Data are presented as means ± SEM from three donors. † and ¥ indicate p<0.05 significance as compared to−epinephrine and −epinephrine + IL-13 treated cells respectively.</p

β₂ARs are required for mucin production in response to IL-13 in NHBE cells.

<p>NHBE cells were grown in the presence of 3 μM epinephrine, then at ALI, they were treated with 20 ng/ml IL-13 in combination with 3 μM CGP-20712A (a preferential β₁AR antagonist) or 1 μM ICI-118,551 (a preferential β₂AR antagonist) for 14 days. <b>A</b>: MUC5AC transcripts were quantified from extracted total RNA by qRT-PCR. Data are presented as fold change compared to cells grown in the presence of epinephrine only. <b>B</b>: Quantification of intracellular mucin 5AC content. The ratio of mucin 5AC integrated density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132559#pone.0132559.s003" target="_blank">S3A Fig</a>). <b>C</b>: Quantification of intracellular mucin glycoproteins in response to different ligands. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to control cells. See the supplement for the representative images (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132559#pone.0132559.s003" target="_blank">S3B Fig</a>). Data are presented as means ± SEM from three donors. *, # and @ indicate p<0.05 significance as compared to + epinephrine, + epinephrine + IL-13 and + epinephrine + IL-13 + CGP-20721A treated cells respectively.</p