β2-Adrenergic agonists attenuate organic dust-induced lung inflammation

Abstract

Agricultural dust exposure results in significant lung inflammation, and individuals working in concentrated animal feeding operations (CAFOs) are at risk for chronic airway inflammatory diseases. Exposure of bronchial epithelial cells to aqueous extracts of hog CAFO dusts (HDE) leads to inflammatory cytokine production that is driven by protein kinase C (PKC) activation. cAMP-dependent protein kinase (PKA)-activating agents can inhibit PKC activation in epithelial cells, leading to reduced inflammatory cytokine production following HDE exposure. β2-Adrenergic receptor agonists (β2-agonists) activate PKA, and we hypothesized that β2-agonists would beneficially impact HDE-induced adverse airway inflammatory consequences. Bronchial epithelial cells were cultured with the short-acting β2-agonist salbutamol or the long-acting β2-agonist salmeterol prior to stimulation with HDE. β2-Agonist treatment significantly increased PKA activation and significantly decreased HDE-stimulated IL-6 and IL-8 production in a concentration- and time-dependent manner. Salbutamol treatment significantly reduced HDE-induced intracellular adhesion molecule-1 expression and neutrophil adhesion to epithelial cells. Using an established intranasal inhalation exposure model, we found that salbutamol pretreatment reduced airway neutrophil influx and IL-6, TNF-α, CXCL1, and CXCL2 release in bronchoalveolar lavage fluid following a one-time exposure to HDE. Likewise, when mice were pretreated daily with salbutamol prior to HDE exposure for 3 wk, HDE-induced neutrophil influx and inflammatory mediator production were also reduced. The severity of HDE-induced lung pathology in mice repetitively exposed to HDE for 3 wk was also decreased with daily salbutamol pretreatment. Together, these results support the need for future clinical investigations to evaluate the utility of β2-agonist therapies in the treatment of airway inflammation associated with CAFO dust exposure.

Keywords: agricultural dusts; bronchial epithelial cells; concentrated animal feeding operations; lung inflammation; β2-agonists.

Fig. 1.

Pretreatment of bronchial epithelial cells (BECs) with β₂-agonists dose-responsively inhibits hog dust extract (HDE)-induced cytokine release. Cultured immortalized human bronchial epithelial (BEAS-2B; A) and primary human bronchial epithelial (NHBE; B) cells were exposed to various concentrations of the long-acting β₂-agonist salmeterol or the short-acting β₂-agonist salbutamol for 1 h and then to 5% HDE in the presence or absence of β₂-agonist for an additional 24 h. Supernatant medium was analyzed for IL-6 and IL-8 levels by ELISA. Values are means ± SE for 3 (A) or 4 (B) independent experiments. *P < 0.05 vs. HDE alone (by 1-way ANOVA and Tukey's posttest).

Fig. 2.

β₂-Agonists significantly attenuate HDE-mediated BEAS-2B cell cytokine release over time. Cultured BEAS-2B cells were treated with salmeterol (A) or salbutamol (B) at 10 μM in the presence or absence of 5% HDE for 2, 6, or 12 h. Supernates were collected for IL-6 and IL-8 measurements by ELISA. Both β₂-agonists significantly diminished the dust-induced release of IL-6 by 12 h and IL-8 as early as 6 h following HDE challenge. Values are means ± SE for 3 parallel experiments (12 technical replicates per condition). *P < 0.05 vs. HDE + salmeterol (A) or HDE + salbutamol (B) (by 1-way ANOVA and Tukey's posttest).

Fig. 3.

Salbutamol and salmeterol stimulate PKA activity in cultured BEAS-2B cells. Subconfluent BEAS-2B monolayers were pretreated with β₂-agonists (10 μM) for 1 h prior to 5% HDE challenge or medium alone for an additional 1 h. Cell lysates were flash-frozen for PKA analysis. Both β₂-agonists significantly induced PKA activity in cell lysates. HDE had no effect on PKA activity either alone or in combination with β₂-agonist. Values are means ± SE of data pooled from 2 independent experiments. *P < 0.05 vs. vehicle, #P < 0.05 vs. HDE alone (by 1-way ANOVA and Tukey's posttest).

Fig. 4.

Pretreatment of epithelial cells with the short-acting β₂-agonist salbutamol significantly dampens the HDE-induced binding of neutrophils. BEAS-2B monolayers were treated with or without 10 μM salbutamol for 1 h and then with 5% HDE in the presence or absence of salbutamol for 24 h. Freshly isolated human neutrophils (0.5 × 10⁶/well) were labeled with calcein-AM and allowed to attach to the treated epithelial cells for 40 min. Unattached PMNs were removed, and adherent cells were quantified by residual fluorescence as percentage of the initial number of PMNs in the coculture. Values are means ± SE derived from 3 parallel experiments (18 technical replicates per condition). *P < 0.05 vs. HDE alone, #P < 0.05 vs. salbutamol alone (by 1-way ANOVA).

Fig. 5.

Dust extract-mediated surface expression of ICAM-1 is significantly diminished when BEAS-2B cells are pretreated with salbutamol. Subconfluent BEAS-2B cultures were incubated with 10 μM salbutamol for 1 h prior to 5% HDE challenge for an additional 24 h. Cells were detached from the plates and probed for surface ICAM-1 using a sensitive and specific phycoerythrin-conjugated monoclonal antibody. A total of 10,000 gated events were captured per condition. A: mean fluorescence intensity histograms from 1 representative experiment. B: mean signal intensity derived from pooling results of 3 experiments. *P < 0.05 vs. HDE alone, #P < 0.05 vs. medium alone (by Student's paired t-test).

Fig. 6.

Nebulized salbutamol pretreatment attenuates HDE-mediated inflammatory cell influx and cytokine release in the airways of acutely exposed mice. Mice (n = 5 per condition) were exposed to nebulized salbutamol in an exposure chamber 30 min before a single challenge with 12.5% intranasal HDE or saline. At 5 h following the HDE exposure, mice were euthanized, and bronchoalveolar lavage was performed. Total cells and differential counts in bronchoalveolar lavage fluid (BALF) were quantified (A), and cytokine concentrations were determined by ELISA (B). CXCL, chemokine C-X-C motif ligand; MIP-2, macrophage inflammatory protein-2; KC, keratinocyte chemoattractant. *P < 0.05 vs. HDE alone (by 1-way ANOVA and Tukey's posttest).

Fig. 7.

HDE-induced ICAM-1-specific immunostaining on airway luminal epithelial cells is abrogated when mice are pretreated with salbutamol. Paraffin-embedded lung sections from mice exposed a single time to 12.5% HDE (B) demonstrate prominent epithelial ICAM-1 immunostaining compared with mice given saline alone (A). Tissues from mice pretreated with nebulized salbutamol and then challenged with HDE showed a marked reduction in ICAM-1 staining (C). Micrographs are representative of lung sections from 3 mice per condition. Arrows indicate localization of ICAM-1 on the apical surface of the epithelium. Parenchymal alveolar cells stained equally positive in all groups.

Fig. 8.

Inflammatory cell recruitment and cytokine/chemokine production are reduced in airways of mice repeatedly exposed to HDE following β₂-agonist treatments. Each day for 15 consecutive weekdays, mice (5 per group) were given nebulized salbutamol 30 min prior to intranasal HDE challenge. At 5 h following the final HDE exposure, mice were euthanized, BALF was collected, and lungs were removed for histological analysis. Inflammatory cells were recovered from BALF and quantified (A), and cytokines were measured in the BALF supernates (B). The entire experiment was performed twice, resulting in 10 mice per condition. *P < 0.05 vs. HDE alone (by 1-way ANOVA and Tukey's posttest).

Fig. 9.

Salbutamol improves the histopathology of mice repeatedly exposed to HDE. Paraffin-embedded hematoxylin-eosin-stained thin sections of inflated lungs removed from mice exposed to HDE for 15 days exhibit both diffuse parenchymal inflammatory infiltrates (E and F) and focal mononuclear aggregates (E and F insets) compared with control mice (A and B) and mice treated with salbutamol alone (C and D). In mice pretreated with salbutamol prior to HDE, inflammatory cell influx is markedly blunted (G and H). The entire experiment was performed twice, resulting in 10 mice per condition. Micrographs are representative of 8 slides per condition. In I, inflammatory scores [from 0 (no inflammation) to 3 (severe inflammation)] were assigned to each treatment group by a clinical pathologist blinded to study parameters. *P < 0.05 vs. HDE alone (by 1-way ANOVA and Bonferroni's posttest).