Alcohol Decreases Organic Dust-Stimulated Airway Epithelial TNF-Alpha Through a Nitric Oxide and Protein Kinase-Mediated Inhibition of TACE

Abstract

Background: Farm workers in rural areas consume more alcohol than those who reside in urban areas. Occupational exposures such as agricultural work can pose hazards on the respiratory system. It is established that hog barn dust induces inflammation in the airway, including the release of cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-8. We have shown that alcohol alters airway epithelial innate defense through changes in both nitric oxide (NO) and cAMP-dependent protein kinase A (PKA). Simultaneous exposure to hog barn dust and alcohol decreases inflammatory mediators, TNF-α, IL-6, and IL-8, in mice. Previously, mice exposed to both alcohol and hog barn dust showed a depleted amount of lymphocytes compared to mice exposed only to hog barn dust. Weakening of the innate immune response could lead to enhanced susceptibility to disease. In addition, mice that were co-exposed to hog barn dust and alcohol also experienced increased mortality.

Methods: Because we recently demonstrated that PKA activation inhibits the TNF-α sheddase, TNF-α-converting enzyme (TACE), we hypothesized that an alcohol-mediated PKA pathway blocks TACE activity and prevents the normative inflammatory response to hog barn dust exposure. To delineate these effects, we used PKA pathway inhibitors (adenylyl cyclase [AC], cAMP, and PKA) to modulate the effects of alcohol on dust-stimulated TNF-α release in the bronchial epithelial cell line, BEAS-2B. Alcohol pretreatment blocked TACE activity and TNF-α release in hog barn dust-treated cells.

Results: Alcohol continued to block hog barn dust-mediated TNF-α release in the presence of the particulate AC inhibitor, SQ22,536. The soluble adenylyl cyclase inhibitor, KH7, however, significantly increased the inflammatory response to hog barn dust. phosphodiesterase 4 inhibitors significantly elevated cAMP and enhanced alcohol-mediated inhibition of dust-stimulated TNF-α release. In addition, the NO synthase inhibitor, l-NMMA, also reversed the alcohol-blocking effect on dust-stimulated TNF-α.

Conclusions: These data suggest that alcohol requires a soluble cyclase-generated cAMP-PKA pathway that is dependent upon the action of NO to inhibit TACE and TNF-α release. These findings support our observations that alcohol functions through a dual NO and PKA pathway in bronchial epithelial cells.

Keywords: Airways; Alcohol; Inflammation; Nitric Oxide; TNF-α-Converting Enzyme.

Figure 1. Alcohol blocks HDE-stimulated PKCε activation and IL-8 release in BEAS-2B

A. BEAS-2B cells were exposed to media, 5% HDE, EtOH (100 mM) 24 hr and HDE and EtOH (24 hr prior to the 6 hour exposure) for 6 hr. Following treatment, IL-8 release was assayed. HDE stimulated IL-8 release compared to the controls, b denotes p<0.0001. Co-treatment of HDE-EtOH decreased IL-8 release significantly when compared to HDE-exposed cells, c denotes p<0.0001. B. BEAS-2B cells were exposed to media, 5% HDE and EtOH (100 mM) for 0,1, 6, and 24 hr. Post-treatment cells were assayed for the PKCε activity. HDE increased the PKCε activity when compared to the controls at 6 hr, b denotes p<0.001. EtOH failed to increase the PKCε activity at any of the time points, p<0.001. One-way ANOVA was conducted with a Tukey post-hoc test.

Figure 2. Alcohol blocks HDE-stimulated PKCε activation in primary HBEC

A. HBECs were exposed to media, 5% HDE, EtOH (100µM) and HDE and EtOH for 6 hr. Ethanol treatment occurred 24 hr prior to the 6-hour exposure. Post-treatment IL-8 release was assayed. HDE stimulated IL-8 release when compared to the controls, b signifies p<0.0001. The co-treatment of HDE and EtOH decreased IL-8 release significantly when compared to the HDE-exposed cells, c signifies p<0.0001. B. HBECs were exposed to media, 5% HDE and EtOH (100µM) for 6 hr. Post-treatment cells were assayed for PKCε activity. HDE increased PKCε activity when compared to the controls, b signifies p<0.001. EtOH decreased PKCε activity significantly, a signifies p<0.001. A one-way ANOVA was conducted with a Tukey post-hoc test.

Figure 3. Alcohol does not block HDE-stimulated PKCα activation in BEAS-2B

A. BEAS-2B cells were exposed to media only, which served as the control, HDE (0.1, 1, 5%) in the presence or absence of EtOH (100 mM) for 6 hr. Post-treatment, cells were assayed for PKCα activity. HDE (1% and 5%) increased PKCα activity significantly compared to media only exposed cells, b denotes p<0.05. A co-exposure of HDE and EtOH did not alter PKCα activity, p >0.05. B. BEAS-2B cells were exposed to media, HDE (0.1, 1, 5%) in the presence or absence of EtOH (100 mM) for 6 hr. Post-treatment, cells were assayed for PKCε activity. HDE (1 and 5%) stimulated an increase of PKCε activity when compared to control cells, b denotes p≤ 0.05. When cells were exposed to HDE and EtOH PKCε activity was decreased, a signifies p<0.05 under a one-way ANOVA and Tukey post-test analysis.

Figure 4. PMA-stimulated PKCε activation is not blocked by alcohol

BEAS-2B cells were exposed to media, 5% HDE, EtOH (100 mM), 5%HDE + EtOH (100 mM), PMA (100 ng/ml) and PMA + EtOH (100 mM) for 6 hr. PKCε activity increased with HDE exposure and co-exposure of HDE and EtOH decreased PKCε activity significantly, b denotes p<0.05. PMA stimulated PKCε activity and co-treatment of PMA and EtOH also stimulated PKCε activity, b also denotes p<0.05 under a one-way ANOVA and Tukey post-hoc test.

Figure 5. HDE-stimulated TACE activity and TNF release is blocked by alcohol

A. BEAS-2B cells were exposed to media, 5% HDE, EtOH (100 mM), 5% HDE + EtOH (100 mM) for 6 hr. HDE stimulated an increase in TNFα release when compared to controls, b signifies p<0.01. The co-exposure of HDE and EtOH resulted in a significant decrease in TNFα release when compared to the HDE alone, c signifies p<0.05. Control and EtOH-treated cells were not significantly different. B. BEAS-2B were subjected to the following treatments: media, 5% HDE, HDE + EtOH (100 mM), EtOH (100 mM) alone, HDE (5%)+Tapi-1 (10 µM) and Tapi-1 alone. TACE activity was increased with HDE exposure, but ameliorated when airway cells were exposed to HDE and alcohol. b signifies the increase of TACE activity with HDE exposure, while c denotes a decrease in TACE activity with a co-exposure of HDE and EtOH, p<0.05 under a one-way ANOVA with a Tukey post hoc analysis.

Figure 6. Rp-cAMPS or H89 allow HDE-stimulated IL-6 in presence of EtOH

A. BEAS-2B cells were treated with the following treatments for 6 hr: media, HDE, HDE + EtOH and HDE + ETOH + inhibitors (Rp-cAMPS (5 µmol) or H-89 (10 µM)). Supernatant fractions were extracted and IL-6 was measured via ELISA. Both the antagonist analog (Rp-cAMPS) and PKA inhibitor (H-89) were capable of fully restoring the HDE-mediated cytokine release that was blunted by alcohol when compared to the co-exposed cultures, d denotes p<0.0001 and e signifies p<0.001 under a one-way ANOVA and Tukey post-hoc test.

Figure 7. KH7 inhibitor, not SQ22,536 allowed HDE-stimulated TNFα/IL-8 in presence of EtOH

A. BEAS-2B were subjected to the following treatments for 6 hr: media, 5% HDE, HDE + EtOH (100 mM) and HDE + EtOH + inhibitor (SQ22,536,50 µM; or KH7, 10µM)). Supernatant fractions were extracted and TNFα, IL-8 and IL-6 were measured via ELISA. The KH7 sAC inhibitor was able to partially restore cytokine production (d signifies p <0.05 compared to b (HDE)) whereas the SQ22, 536 pAC inhibitor was not different from the co-exposed (HDE + EtOH) group cultures. B. BEAS-2B cells were exposed to media, HDE, EtOH and HDE with or without IBMX (0.2 mmol/L), Rolipram (8 µM), Ro 20–1724 (20µM). HDE increased TNFα release while EtOH decreased HDE-induced TNFα release b signifies p<0.05 compared to media (a). Treatment with PDE inhibitors resulted in decreased TNFα release, c denotes p<0.05 compared to b. Significance was determined under a one-way ANOVA and Tukey post-hoc test.

Figure 8. L-NMMA allows HDE-stimulated TNFα/IL-8 in presence of EtOH

BEAS-2B were subjected to the following treatments for 6 hr: media, 5% HDE, HDE + EtOH (100 mM) and HDE + ETOH + L-NMMA (100 mM). Supernatant fractions were extracted and pro-inflammatory cytokines TNFα, IL-8 and IL-6 were measured via ELISA. L-NMMA restored HDE-mediated cytokine production compared to HDE-exposed cell cultures, b denotes p>0.05 under a one-way ANOVA and Tukey post-hoc analysis.

Figure 9. Ethanol does not decrease TNF-α levels via NF-κB expression

BEAS-2B cells were incubated with 100 mM ethanol 24 hr followed by a 6-hour treatment of 5% HDE and ethanol. NF-κB activation was assayed as described and expressed as a fold change activation over medium-only treated cells. Media and ethanol were not significantly different. a denotes p>0.05, whereas HDE and HDE and ethanol significantly increased NF-κB expression (b signifies p-value<0.05) under a one-way ANOVA and Tukey post-hoc test.

Figure 10. Summary model diagram

Proposed model of alcohol’s effect on HDE-stimulated pro-inflammatory cytokine release. Alcohol stimulates both nitric oxide (NO) and adenynyl cyclase, which leads to the activation cAMP and subsequent PKA activation, which then inhibits TACE activity. By blunting TACE activity, the HDE-stimulated release of pro-inflammatory cytokines TNFα, IL-8 and IL-6 are decreased. PKCα is activated by dust upstream of TACE and independent of TACE action, while PKCε activity is regulated by dust downstream of TACE and TNFα.