Alcohol Inhibits Organic Dust-induced ICAM-1 Expression on Bronchial Epithelial Cells

Abstract

Aims: Exposure to dusts/bioaerosols in concentrated animal feeding operations (CAFOs) results in inflammatory lung diseases in workers. Hog CAFOs dust extract (HDE) increases expression of intercellular adhesion molecule-1 (ICAM-1), neutrophil adhesion, and TNFα release in bronchial epithelial cells. Alcohol consumption is increasingly recognized to impair lung immunity. We hypothesized that alcohol impairs HDE-induced TNFα, ICAM-1 expression and neutrophil adhesion by directly inhibiting TNFα converting enzyme (TACE) activity.

Methods: Bronchial epithelial cells (BEAS-2B) and primary human bronchial epithelial cells were pretreated with ethanol (EtOH) or TACE inhibitor. ICAM-1 surface expression, TNFα release, and TACE activity were analyzed following HDE stimulation. The effect of alcohol and TACE inhibition on HDE-regulated epithelial cell/neutrophil adhesion interactions was investigated. Finally, utilizing an established animal model, C57BL/6 mice were fed ad libitum ethanol (20%) in drinking water for 8 wk followed by daily intranasal inhalation of HDE or saline during the final two weeks. Mice were sacrificed and lung sections immunostained for ICAM-1.

Results: Pretreatment with alcohol or TACE inhibitor significantly decreased HDE-induced ICAM-1 expression and TNFα release. HDE augmented neutrophil adhesion to epithelial cells, which was decreased with alcohol (32% decrease) or TACE inhibitor (55% decrease) pretreatment. TACE activity increased following HDE exposure, but TACE activity was inhibited following alcohol pretreatment. Alcohol-fed mice demonstrated decreased HDE-induced airway epithelium ICAM-1 expression.

Conclusions: Alcohol diminishes HDE-induced ICAM-1 expression, TNFα release, and neutrophil adhesion via inhibition of TACE activity. These results suggest that alcohol may be an important modulator of lung innate immune responses following CAFO exposure.

Keywords: ICAM-1; alcohol; bronchial epithelial cells; inflammatory lung dieseas; organic dust.

Figure 1. Ethanol dose-dependently activates PKA and inhibits HDE-induced cytokine release from BEAS-2B cells

BEAS-2B were pretreated with various concentrations of EtOH (12.5 to 150 mM) for 1 h before being exposed to 5% HDE (± EtOH) for an additional 24 h. EtOH dose-dependently increases PKA activity at 1 hr (A; black bars) and decreases HDE-stimulated PKC epsilon activity at 6 hr (A; gray bars). Inflammatory cytokines TNFα (B); IL-6 (C); IL-8 (D) measured in culture supernates by ELISA. ^ap<0.05 vs 0 mM EtOH for PKA; ^bp<0.05 vs 0 mM EtOH for PKCε; *p<0.05 vs HDE alone for n=3 or 4 independent experiments (ANOVA, Tukey’s post test).

Figure 2. Alcohol pretreatment down regulates HDE-mediated ICAM-1 expression in human bronchial epithelial cells

BEAS-2B (B) and HBEC (C) were treated with control medium or 5% HDE +/− 100 mM ethanol (EtOH) for 24 hr and FACS analysis was performed. (A) Representative histogram showing significant rightward shift following HDE exposure (dark gray histogram), where open histograms represent isotype control antibody. Panel on far right depicts HDE treatment (dark gray) compared to EtOH+HDE treatment (light gray). Bar graphs depict means with standard error bars from N=6 (B) and N=3 (C) independent experiments. *p<0.05 vs. control medium, ^#p<0.05 vs. HDE.

Figure 3. Alcohol inhibits HDE-induced peripheral blood neutrophil adhesion to epithelial cells

(A) BEAS-2B cells were pretreated with 100 mM ethanol (EtOH) prior to exposure to 5% HDE or control medium for 24 hr. Neutrophils were then allowed to adhere to stimulated BEAS-2B for 30 min. HDE causes a significant increase in PMN adhesion compared to medium alone. Pretreatment with EtOH causes a significant decrease in HDE induced adhesion. (B) Neutrophils were treated directly with 5% HDE and then incubated with BEAS-2B. Treatment of neutrophils with HDE does not stimulate adhesion to untreated BEAS-2B. Bar graphs represent means with standard error bars of 4 independent experiments, *p<0.05 vs. control medium, ^#p<0.05 vs. HDE.

Figure 4. Alcohol blocks ICAM-1 localization in airway epithelium of mice instilled with HDE

Mice were fed either water (control) or 20% ethanol (EtOH) for 8 wk with or without intranasal instillation of HDE. Saline inhalation in either control (A) or EtOH-fed mice (B) exhibit minimal airway epithelial ICAM-1 staining in luminal cells. Mice inhaling HDE (C) show prominent ICAM-1 staining (brown) on apical regions of the luminal airway epithelium and extensive mononuclear cell aggregates (purple). Ethanol pretreatment (D) blocks the HDE-stimulated upregulation of localized ICAM-1. Images are representative of sections from at least three mice per treatment group.

Figure 5

A–C: Pretreatment with TACE inhibitor (TAPI-1) attenuates HDE-mediated ICAM-1 expression (A), PMN adhesion (B), and TNFα release (C) in human bronchial epithelial cells. BEAS-2B cells were treated with control medium, or 5% HDE with or without 20 µM TAPI-1 for 24 hr and FACS for ICAM-1, PMN adhesion assays, and TNFα ELISA on supernates were performed. Bar graphs represent mean values with standard error bars (N=minimum of 3 independent experiments), *p<0.05 vs. control medium, #p<0.05 vs. HDE. D–E: Pretreatment with TAPI-1 dose-dependently decreases HDE-induced cytokine release from BEAS-2B. BEAS-2B were treated with culture medium alone, 5% HDE, 20 µM TACE inhibitor TAPI-1 alone, or were pretreated with various doses of TAPI-1 for 1 hr before exposure to 5% HDE in the presence of TAPI-1 for 24 hr. Conditioned supernatant media were assayed for IL-6 (panel D) and IL-8 (panel E) by ELISA. Bar graphs represent the means with standard error bars of 3 independent experiments (* p<0.05 vs. HDE alone).

Figure 6. Alcohol pre-treatment inhibits HDE-induced TACE activity in BEAS-2B and primary human bronchial epithelial cells

Lysates from BEAS-2B (panel A) primary human bronchial epithelial cells (HBECs; panel B) pretreated with or without 100 mM ethanol for 1 hr before exposure to 5% HDE or control medium for an additional 2, 8 or 20 hr were assayed for TACE activity. HDE stimulated TACE from 2-20 hr, but alcohol pretreatment blunted HDE-induced TACE activity at all time points. Data shown represent the means (+/− SEM) of 3 separate experiments (* p<0.05 vs. HDE+EtOH).

Figure 7. Alcohol does not directly affect TNFα-induced ICAM-1 expression

BEAS-2B were treated with either TNFα (0.1 ng/ml) or TNFα incubated with 100 mM ethanol for 1 hr before epithelial cell treatment. FACS for ICAM-1 revealed a significant increase in ICAM-1 expression even when cells were treated with TNFα + ethanol. Bar graphs represent the mean with standard error bars of 3 separate experiments (* p<0.05 vs. control medium).