Electronic cigarette exposure triggers neutrophil inflammatory responses

Abstract

Background: The use of electronic cigarettes (e-cigs) is increasing and there is widespread perception that e-cigs are safe. E-cigs contain harmful chemicals; more research is needed to evaluate the safety of e-cig use. Our aim was to investigate the effects of e-cigs on the inflammatory response of human neutrophils.

Methods: Neutrophils were exposed to e-cig vapour extract (ECVE) and the expression of CD11b and CD66b was measured by flow cytometry and MMP-9 and CXCL8 by ELISA. We also measured the activity of neutrophil elastase (NE) and MMP-9, along with the activation of inflammatory signalling pathways. Finally we analysed the biochemical composition of ECVE by ultra-high performance liquid chromatography mass spectrometry.

Results: ECVE caused an increase in the expression of CD11b and CD66b, and increased the release of MMP-9 and CXCL8. Furthermore, there was an increase in NE and MMP-9 activity and an increase in p38 MAPK activation. We also identified several harmful chemicals in ECVE, including known carcinogens.

Conclusions: ECVE causes a pro-inflammatory response from human neutrophils. This raises concerns over the safety of e-cig use.

Keywords: COPD; Electronic cigarettes; Inflammation; MMP-9; Neutrophils; Smoking.

Fig. 1

E-cig induced neutrophil activation. Neutrophils from three healthy subjects were exposed to ECVE (0.001–0.1 OD) for 2, 4 or 6 h (white, grey and black bars respectively). Neutrophils were analysed for CD11b (a) and CD66b (b) expression, and shape change (c) by flow cytometry. Data presented as mean ± SEM where *, ** and *** = significant increase compared to unstimulated control (p < 0.05, p < 0.01, and p < 0.001 respectively)

Fig. 2

E-cig induced protease and CXCL8 release. Neutrophils from healthy subjects were exposed to ECVE (0.001–0.1 OD) for 6 h and supernatants were analysed for MMP-9 (a) and CXCL8 (d) release by ELISA (n = 10) and NE activity by florescence (e; n = 6). Culture supernatants from healthy subjects (0.003 ECVE) were also analysed for MMP-9 activity by zymography and MMP-9 expression by western blot (representative image; (b)). Western blots were analysed by densitometry (c). Data presented as mean ± SEM where *, ** and *** = significant change compared to unstimulated control (p < 0.05, p < 0.01, and p < 0.001 respectively)

Fig. 3

E-cig induced cell signalling pathway activation. Neutrophils from five healthy subjects were exposed to ECVE (0.003 and 0.01 OD) for 30 or 60 mins and cell lysates were analysed for a phospho-p38 MAPK, b phospho-ERK and c phospho-p65. All blots were analysed by densitometry and any changes were relative to total p38 MAPK. Data presented as mean ± SEM where * = significant increase compared to unstimulated control (p < 0.05)

Fig. 4

Inhibition of e-cig induced MMP-9 release. Neutrophils from six healthy subjects were pre-incubated with dexamethasone (dex; 1 μM), selumetinib (Sel; 1 μM) or BIRB-796 (1 μM) for 1 h prior to ECVE (0.003) exposure for 6 h. Supernatants were analysed for MMP-9 by ELISA. Data presented as mean ± SEM where * = significant increase compared to unstimulated control (p < 0.05)

Fig. 5

E-cig induced MMP-9 release; brand comparisons. Neutrophils from ten healthy subjects were exposed to brand 1 (0 or 24 mg; (a and d)), brand 2 (0 or 24 mg; (b and e)), brand 3 (16 mg; (c)), or CSE (f) for 6 h. Supernatants were analysed for MMP-9 by ELISA. Data presented as mean ± SEM where *, ** and *** = significant increase compared to unstimulated control (p < 0.05, p < 0.01, and p < 0.001 respectively)

Fig. 6

E-cig induced CXCL8 release; brand comparisons. Neutrophils from ten healthy subjects were exposed to brand 1 (0 or 24 mg; (a and d)), brand 2 (0 or 24 mg; (b and e)), brand 3 (16 mg; (c)), or CSE (f) for 6 h. Supernatants were analysed for CXCL8 by ELISA. Data presented as mean ± SEM where *, ** and *** = significant increase compared to unstimulated control (p < 0.05, p < 0.01, and p < 0.001 respectively)

Fig. 7

Acrolein induced MMP-9 release. Neutrophils from six healthy subjects were exposed to acrolein (0.1–1 μM) for 6 h. Supernatants were analysed for MMP-9 by ELISA. Data presented as mean ± SEM where * = significant increase compared to unstimulated control (p < 0.05)