Inflammatory and cytotoxic effects of acrolein, nicotine, acetylaldehyde and cigarette smoke extract on human nasal epithelial cells

Abstract

Background: Cigarette smoke induces a pro-inflammatory response in airway epithelial cells but it is not clear which of the various chemicals contained within cigarette smoke (CS) should be regarded as predominantly responsible for these effects. We hypothesised that acrolein, nicotine and acetylaldehyde, important chemicals contained within volatile cigarette smoke in terms of inducing inflammation and causing addiction, have immunomodulatory effects in primary nasal epithelial cell cultures (PNECs).

Methods: PNECs from 19 healthy subjects were grown in submerged cultures and were incubated with acrolein, nicotine or acetylaldehyde prior to stimulation with Pseudomonas aeruginosa lipopolysaccharide (PA LPS). Experiments were repeated using cigarette smoke extract (CSE) for comparison. IL-8 was measured by ELISA, activation of NF-κB by ELISA and Western blotting, and caspase-3 activity by Western blotting. Apoptosis was evaluated using Annexin-V staining and the terminal transferase-mediated dUTP nick end-labeling (TUNEL) method.

Results: CSE was pro-inflammatory after a 24 h exposure and 42% of cells were apoptotic or necrotic after this exposure time. Acrolein was pro-inflammatory for the PNEC cultures (30 μM exposure for 4 h inducing a 2.0 fold increase in IL-8 release) and also increased IL-8 release after stimulation with PA LPS. In contrast, nicotine had anti-inflammatory properties (0.6 fold IL-8 release after 50 μM exposure to nicotine for 24 h), and acetylaldehyde was without effect. Acrolein and nicotine had cellular stimulatory and anti-inflammatory effects respectively, as determined by NF-κB activation. Both chemicals increased levels of cleaved caspase 3 and induced cell death.

Conclusions: Acrolein is pro-inflammatory and nicotine anti-inflammatory in PNEC cultures. CSE induces cell death predominantly by apoptotic mechanisms.

Figure 1

Immunocytochemistry for cytokeratin 5 in nasal epithelial cell cultures. Primary nasal epithelial cell cultures were stained with a rabbit anti-human antibody against cytokeratin 5 (1:100). The primary antibody was detected using a secondary antibody coupled to Alexafluor 568 (1:500). Nuclei were stained blue with DAPI (×40). Each quadrant represents DAPI staining alone (upper left), staining for cytokeratin 5 alone (upper right), and staining for both DAPI and cytokeratin 5 (lower left).

Figure 2

IL-8 release from PNECs after 24 h treatment with PA LPS in the presence or absence of 30 μM acrolein for 1 h or 4 h, 50 μM nicotine or 50 μM acetylaldehyde for 4 h or 24 h, and 5% CSE for 4 h or 24 h. PNEC cultures were treated with increasing concentrations of LPS for 24 h ± pretreatment with acrolein, nicotine or acetylaldehyde (n = 4). Separate cultures were treated with 5% CSE for 4 h or 24 h (n = 4). Data are displayed as median ± IQR. * p < 0.05. The control IL-8 concentration was 1310 ± 223 pg/ml.

Figure 3

Effect of acrolein and nicotine on caspase-3 activation in PNEC cultures determined using western blotting. Western blots of cleaved caspase 3 in healthy PNECs (with beta-actin loading controls). Blot A: lanes 1–3 represent treatment of PNECs with 0, 5, 50 μM nicotine (1 h); blot B: lanes 1–3: 0, 10, 50 μM acrolein (1 h); blot C: lanes 1–3 represent treatment of PNECs with 0, 5, 50 μM acetylaldehyde (1 h) and blot D lanes 1–3: 0%, 5%, 50% CSE (1 h) respectively.

Figure 4

Effect of acrolein, nicotine, acetylaldehyde and CSE treatment on cell viability. In each plot, the horizontal axis represents intensity of staining for Annexin V and vertical axis intensity of staining for PI (determined in the FL1 and FL3 plot respectively, both logarithmic scale). Dot plots represent (A) untreated cells, (B) cells treated with Triton-X for 4 h, (C) cells treated with 5 μM staurosporin for 4 h, (D) cells treated with media alone for 4 h, (E) cells treated with 50 μM nicotine for 24 h, (F) cells treated with 50 μM acrolein for 4 h, (G) cells treated with 50 μM acetylaldehyde for 24 h, and (H) cells treated with 5% CSE for 24 h.

Figure 5

Effect of acrolein, nicotine, acetylaldehyde and CSE on apoptosis in PNEC cultures using TUNEL assay. Nasal epithelial cells were grown on coverslips and treated with (A) PBS or (B) DNase I solution for the negative and positive control respectively. Separate samples were treated with (C) 50 μM acrolein for 4 h, (D) 50 μM nicotine for 24 h, (E) 50 μM acetylaldehyde for 24 h or (F) 5% CSE for 24 h. Levels of apoptosis were determined using the Click-iT reaction according to manufacturer’s instructions.

Figure 6

Effect of acrolein, nicotine and acetylaldehyde on NF-κB expression in PNEC cultures determined using western blotting. Western blot of phosho-NF-κB and IκB-α protein expression in PNECs (with beta-actin loading controls). Blot A: lanes 1–3 represent treatment of PNECs with 0, 10, 30 μM acrolein (1 h); blot B: lanes 1–3: 0, 5, 50 μM nicotine (1 h); blot C: lanes 1–3 represent treatment of PNECs with 0, 10, 50 μM acetylaldehyde (1 h) and blot D lanes 1–3: media, 5% CSE, 50% CSE (1 h). Densitometry included for phosho-NF-κB: actin ratio (n = 5 for each group). Data are displayed as median ± IQR. *p < 0.05.

Figure 7

Effect of acrolein, nicotine and acetylaldehyde on NF-κB p65 activation in PNEC cultures determined using the TransAM NF-κB p65 kit. Nuclear extracts from PNEC cultures after treatment with 0, 10, 30 μM acrolein (1 h), 0, 5, 50 μM nicotine (1 h), 0, 10, 50 μM acetylaldehyde (1 h) or media, 5% CSE or 50% CSE (1 h) were assayed for NF-κB p65 activation using the TransAM NF-κB p65 Kit (n = 5). Data are displayed as median ± IQR. *p < 0.05.