Co-exposure to cigarette smoke and alcohol decreases airway epithelial cell cilia beating in a protein kinase Cε-dependent manner

Abstract

Alcohol use disorders are associated with increased lung infections and exacerbations of chronic lung diseases. Whereas the effects of cigarette smoke are well recognized, the interplay of smoke and alcohol in modulating lung diseases is not clear. Because innate lung defense is mechanically maintained by airway cilia action and protein kinase C (PKC)-activating agents slow ciliary beat frequency (CBF), we hypothesized that the combination of smoke and alcohol would decrease CBF in a PKC-dependent manner. Primary ciliated bronchial epithelial cells were exposed to 5% cigarette smoke extract plus100 mmol/L ethanol for up to 24 hours and assayed for CBF and PKCε. Smoke and alcohol co-exposure activated PKCε by 1 hour and decreased both CBF and total number of beating cilia by 6 hours. A specific activator of PKCε, DCP-LA, slowed CBF after maximal PKCε activation. Interestingly, activation of PKCε by smoke and alcohol was only observed in ciliated cells, not basal bronchial epithelium. In precision-cut mouse lung slices treated with smoke and alcohol, PKCε activation preceded CBF slowing. Correspondingly, increased PKCε activity and cilia slowing were only observed in mice co-exposed to smoke and alcohol, regardless of the sequence of the combination exposure. No decreases in CBF were observed in PKCε knockout mice co-exposed to smoke and alcohol. These data identify PKCε as a key regulator of cilia slowing in response to combined smoke and alcohol-induced lung injury.

Figure 1

Smoke and alcohol slow cilia beat frequency (CBF). Primary ciliated bovine bronchial epithelial cells were treated with 5% cigarette smoke extract (CSE) and 100 mmol/L ethanol (EtOH) in liquid submerged in vitro cultures. CBF (A) and the average number of motile points per field of cells (B) were determined by Sisson-Ammons Video Analysis. A representative media control (M199) is indicated by the white bar. Data are shown as means ± SEs (n = 9). *P < 0.05 versus control media at matched time points of 3 to 6 hours for CBF; ^†P < 0.01 versus control media at matched time points of 6 to 24 hours for average motile points.

Figure 2

Effect of combination smoke and alcohol on protein kinase C (PKC)ε activity. Primary ciliated bovine bronchial epithelial cells were treated with 5% cigarette smoke extract (CSE) and 100 mmol/L ethanol (EtOH) in submerged in vitro cultures. PKCε activity was assayed at various time points from 30 minutes to 24 hours. A representative media control (M199) is indicated by the white bar. Data are shown as means ± SEs (n = 9). *P < 0.001 versus control media at matched time points of 1 to 3 hours; ^†P < 0.05 versus control media at matched time points of 6 to 9 hours.

Figure 3

Effect of a protein kinase C (PKC)ε activator on cilia beat. Primary ciliated bovine bronchial epithelial cells were treated with 10 μmol/L 8-[2-(2-pentylcyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) for 30 minutes to 24 hours in submerged in vitro cultures. Ciliary beat frequency (CBF; A) was determined by Sisson-Ammons Video Analysis, and PKCε activity was determined for various concentrations (10 nmol/L to 10 μmol/L) of DCP-LA (B) and at various times (15 minutes to 24 hours) of 10 μmol/L DCP-LA (C). Media controls (M199) are indicated by white bars. Data are shown as means ± SEs (n = 9). *P < 0.01 versus control media at matched time points of 2 to 24 hours for CBF; ^†P < 0.05 versus control media at 30 minutes for 100 nmol/L to 10 μmol/L DCP-LA for PKCε activation; ^‡P < 0.05 versus control media at matched time points of 15 to 30 minutes for 10 μmol/L DCP-LA for PKCε activation.

Figure 4

Differential effects of smoke and alcohol on basal versus ciliated cells. Both nonciliated basal (A) and ciliated (B) primary bovine bronchial epithelial cells were treated in submerged culture with M199 media (white bars), 5% cigarette smoke extract (CSE), 100 mmol/L ethanol (EtOH), individually and in combination for 1 hour, and protein kinase C (PKC)ε activity assayed. As a positive control, cells were treated with 100 ng/mL phorbol-12-myristate-13-acetate (PMA) for 15 minutes, and PKCε activity was assayed. Data are shown as means ± SEs (n = 9). *P < 0.001 versus control media for PMA treatment in both cell types and smoke+EtOH in ciliated cells.

Figure 5

Differential effects of translocation inhibitor versus catalytic site inhibitor on ciliated cell protein kinase C (PKC)ε and motility. Ciliated primary bovine bronchial epithelial cells were treated with 10 μmol/L myristolated PKCε translocation inhibitor peptide in the presence or absence of 50 μmol/L digitonin (black bars) or 10 μmol/L active site inhibitor Ro 31-8220 and PKCε activity at 2 hours (A) or number of motile points from 1 to 6 hours (B) assayed. Data are shown as means ± SEs (n = 9). *P < 0.001 versus control media for Ro 31-8220 treatment. CBF, ciliary beat frequency.

Figure 6

Localization of protein kinase C (PKC)ε directly on the isolated bovine trachea ciliary axoneme. Axonemes were visualized by differential interference contrast microscopy (A), stained with rabbit anti-PKCε antibodies (B) or nonspecific IgG (C), and visualized by confocal laser scanning microscopy. Axonemes were also assayed for PKCε activity in the presence or absence of calcium, lipid, substrate, or dithiothrietol (DTT) (D).

Figure 7

Effect of combination smoke and alcohol on cilia in lung slices. Precision-cut mouse lung slices were treated with 5% cigarette smoke extract (CSE) and 100 mmol/L ethanol (EtOH) in submerged in vitro culture. Ciliary beat frequency (CBF; A) and the average number of motile points per field of cells (B) were determined by Sisson-Ammons Video Analysis from 1 to 21 hours. Protein kinase C (PKC)ε activity was assayed from 1 to 6 hours (C). Representative media controls (M199) are indicated by the white bars. Data are shown as means ± SEs (n = 9). *P < 0.05 versus control media at matched time points of 3 to 21 hours for CBF; ^†P < 0.01 versus control media at matched time points of 4 to 21 hours for average motile points; ^‡P < 0.001 versus control media at matched time points of 1 to 3 hours.

Figure 8

Effect of in vivo smoke and alcohol exposure on cilia. Mice were either sham-treated (white bars) with air and water, whole body exposed to cigarette smoke (smoke), fed 20% alcohol (EtOH), or exposed to both alcohol and cigarette smoke in combination (smoke+EtOH) for 8 weeks. Ciliary beat frequency (CBF; A) and protein kinase C (PKC)ε activity (B) were assayed from tracheal epithelium. Data are shown as means ± SEs (n = six mice per group). *P < 0.01 for smoke+EtOH-treated versus sham-treated mice.

Figure 9

Sequence of in vivo smoke and alcohol co-exposure does not alter the effects on protein kinase C (PKC)ε. Mice were either whole-body smoke-exposed first followed by alcohol feeding (gray bars) or alcohol-fed first followed by whole-body smoke exposure (black bars) in vivo, and PKCε activity was measured in both tracheal epithelium (A) and precision-cut lung slices (B). Data are shown as means ± SEs (n = 6 mice/group). *P < 0.01 for either smoke then EtOH-treated or EtOH then smoke-treated versus sham-treated mice.

Figure 10

Effect of smoke and alcohol exposure on cilia from protein kinase C (PKC)ε knockout mice. Tracheal rings and lung slices were cut from mice that lacked PKCε expression. Change in ciliary beat frequency (CBF) in response to ex vivo 10 μmol/L Ro 31-8220 treatment (versus baseline CBF) in the trachea of wild-type, PKCε knockout (PKCεKO), and PKCδ knockout (PKCδ-KO) mice were assayed by Sisson-Ammons Video Analysis (A). Lung slice PKCε activity in response to ex vivo treatment with 10 μmol/L 8-[2-(2-pentylcyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) was assayed in wild-type (WT) and PKCεKO mice (B). Lung slice PKCε activity in response to in situ treatment with smoke and alcohol was assayed in WT and PKCεKO mice (C). Data are shown as means ± SEs (n = 6). *P < 0.01 for changes in CBF in WT and PKCδKO mice in the presence versus absence of Ro 31-8220; ^†P < 0.001 for PKCε activation in WT versus PKCεKO mice in response to DCP-LA; ^‡P < 0.001 for PKCε activation in WT versus PKCεKO mice in response to the combination of smoke+alcohol. EtOH, ethanol.