β2AR signalling in normal human bronchial epithelial cells is required for interleukin-13 induced expression of the mucin MUC5AC and the secretion of proinflammatory chemokines.

Indira Pokkunuri1, Nour Al-Sawalha1, Ozozoma Omoluabi1, Long Nguyen2, Burton Dickey2, Richard Bond1, Brian Knoll1. 1University of Houston, Houston, TX, USA, 2University of Texas MD Anderson Cancer Center, Houston, TX, USA

In previous studies, we found that β2-adrenoceptor (β2AR) signalling stimulates the expression of an asthma-like phenotype in mice sensitized and challenged with antigen, or treated intranasally with interleukin-13 (IL-13), a T-helper 2 cytokine implicated in asthma pathogenesis. We also obtained evidence that in mice, airway epithelial cells were key players in mediating the effect of β2AR signalling on airway inflammation. To further delineate the role of airway epithelium in asthma and to confirm these findings in a human model, we used commercially obtained (Lonza) normal human bronchial epithelial (NHBE) cells grown as monolayers on Transwell filter inserts until the cells reached confluence at the air-liquid interface (ALI). The expression of a mRNA encoding a mucin glycoprotein (MUC5AC) was measured in cells cultured for 14 days at ALI with (1) no epinephrine and no IL-13; (2) plus 2 μM epinephrine only; (3) plus 20 ng/ml IL-13 only; (4) epinephrine plus IL-13; and (5) epinephrine plus IL-13 and nadolol at 2 μM. Total RNA was harvested and assayed using quantitative RT-PCR to measure the abundance of MUC5AC transcripts normalized to 18S RNA. Cells from three individual donors were cultured and analyzed (N = 3). The abundance of MUC5AC transcripts was very low when the NHBE cells were cultured without epinephrine (0.77±0.5 copies/105 copies 18S RNA), or with epinephrine alone (1.18±0.7 copies) or IL-13 alone (0.68±0.36 copies). (Numbers are mean values ± standard deviation). MUC5AC transcript levels increased 14.9-fold in cells cultured with IL-13 plus epinephrine (17.63±4.2 copies) compared with epinephrine alone, and the inclusion of nadolol reduced MUC5AC transcript levels back to near basal levels (1.43±1.2 copies; P = 0.0006 comparing +/- nadolol, one-way t-test). In additional studies, we measured the secretion of a subset of chemokines known to be expressed by cultured NHBE cells, comparing cells cultured with (1) epinephrine alone; (2) epinephrine plus IL-13; and (3) epinephrine plus IL-13 and nadolol. Culture subnatants (containing basolateral secretions) were collected between 3 and 5 days after the establishment of ALI, and chemokine concentrations were measured by multiplex ELISA. Epinephrine plus IL-13 treatment increased the concentrations (in pg/ml) of following chemokines over epinephrine alone: the eosinophil attractant eotaxin-2 (from 14.7±6.1 to 101.1± 6.3, a 6.8x increase); CXCL-1, a neutrophil attractant (1467±15 to 5631±317, a 3.84x increase), and MCP-1, an attractor of monocytes, dendritic cells and memory T-cells (4.6±1 to 31.9±1.4, a 6.95x increase) (P<0.001,one way ANOVA, Tukey’s post test). Cells treated with epinephrine plus IL-13 and nadolol secreted near basal levels of these chemokines (eotaxin-2, 6.9±2.2; CXCL1, 1791±276; MCP-1, 14.2±1.9)(P<0.001,one way ANOVA, Tukey’s post test). (Numbers are mean values ± standard deviation). The secretions of CCL-1 (a monocyte attractor) and MIP-3α (a lymphocyte and dendritic cell attractor) were unaffected by IL-13 treatment. Our findings provide strong evidence that β2AR signalling promotes both mucous production and the secretion of proinflammatory chemokines from human airway epithelium, and that beta-blocking drugs may have some utility as anti-inflammatory agents in asthma.