Exposure to biomass smoke extract enhances fibronectin release from fibroblasts

Abstract

COPD induced following biomass smoke exposure has been reported to be associated with a more fibrotic phenotype than cigarette smoke induced COPD. This study aimed to investigate if biomass smoke induced extracellular matrix (ECM) protein production from primary human lung fibroblasts in vitro. Primary human lung fibroblasts (n=5-10) were stimulated in vitro for up to 72 hours with increasing concentrations of biomass smoke extract (BME) or cigarette smoke extract (CSE) prior to being assessed for deposition of ECM proteins, cytokine release, and activation of intracellular signalling molecules. Deposition of the ECM proteins perlecan and fibronectin was upregulated by both CSE (p<0.05) and BME (p<0.05). The release of the neutrophilic chemokine IL-8 was also enhanced by BME. ERK1/2 phosphorylation was significantly upregulated by BME (p<0.05). Chemical inhibition of ERK signalling molecules partially attenuated these effects (p<0.05). Stimulation with endotoxin had no effect. This study demonstrated that BME had similar effects to CSE in vitro and had the capacity to directly induce fibrosis by upregulating production of ECM proteins. The mechanisms by which both biomass and cigarette smoke exposure cause lung damage may be similar.

Figure 1. Biomass smoke and cigarette smoke have similar particle consistency.

Quantity and distribution of particles from the smoke of one cigarette (A) or one unit of biomass (B) as measured by laser particle counter. Data expressed as particles per cubic metre or as % of total particle counts (C–D) for particles in the size ranges of 0.3–0.5, 0.5–1, 1–5, 5–10, 10–25 or <25 uM diameter. Bars represent mean ± SEM. Each experiment was performed in triplicate.

Figure 2. High concentrations of biomass smoke are cytotoxic.

Assessment of cytotoxicity of biomass smoke extract (BME) to human lung fibroblasts (n = 3) as measured by LDH assay (A), MTT assay (B) or manual cell counts (C) following stimulation with 5% cigarette smoke extract (CSE) or 1, 5, or 20% BME in 0.1% FBS/DMEM. Data expressed as % of unstimulated. Bars represent mean ± SEM. Data analysed by one-way ANOVA with Dunnet's post test.*p<0.05 vs unstimulated, n = 6.

Figure 3. Biomass smoke extract enhances deposition of fibronectin from fibroblasts.

Deposition of fibronectin (n = 16) (A) or perlecan (n = 6) (B) from human lung fibroblasts as measured by ECM ELISA following 72 hours stimulation with 5% cigarette smoke extract (CSE) or 1, 5, 10 or 20% biomass smoke extract (BME) in 0.1% FBS/DMEM. Data expressed as absorbance at 405 nm with media alone baseline removed (A,B). Bars represent mean ± SEM. Data analysed by one-way ANOVA with Dunnet's post-test.*p<0.05 vs unstimulated, n = 6–16.

Figure 4. Chemical inhibition of the ERK signalling pathway attenuates biomass smoke induced fibronectin deposition.

Fibroblasts were pretreated for 1(10 µM) (A,B) or UO126 (5 µM) (C,D) in DMSO (vehicle control) before stimulation with 10% (A,C) and 20% BME (B,D) in the presence of inhibitors for 72 hours, prior to analysis of fibronectin deposition by ECM ELISA. Data expressed as % of unstimulated. Bars represent mean ± SEM. Data analysed by T-Test. *p<0.05 vs vehicle control, n = 5.

Figure 5. Biomass smoke exposure upregulates release of IL-8 from fibroblasts.

Release of interleukin (IL)-8 from human lung fibroblasts (n = 10) in response to 72 hours stimulation with 1, 5, 10 or 20% biomass smoke extract (BME) in 0.1% FBS/DMEM as measured by IL-8 ELISA. Data expressed as pg/ml. Bars represent mean ± SEM. Data analysed by one-way ANOVA with Dunnet's post-test. *p<0.05 vs unstimulated, n = 10.

Figure 6. Chemical inhibition of the ERK signalling pathway attenuates BME induced IL-8 release.

Fibroblasts were pretreated for 1(1, 10 µM) or UO126 (0.5, 5 µM) in DMSO (vehicle control) before stimulation with 10% (A) and 20% (B) BME in the presence of inhibitors for 72 hours, prior to analysis of IL-8 release by ELISA. Data expressed as pg/ml. Bars represent mean ± SEM. Data analysed by one-way ANOVA with Bonferroni's post-test.*p<0.05 vs vehicle control, n = 5.

Figure 7. BME activates ERK1/2 signalling molecules.

Fibroblasts were stimulated for 2% CSE or 1, 5, 10 or 20% BME in 0.1% FBS/DMEM, before whole cell lysates were collected and ERK1 (A) or ERK/2 (B) phosphorylation was assessed by western blotting. Data expressed as the ratio of pERK over GAPDH (housekeeping protein). Bars represent mean ± SEM. Data analysed by one-way ANOVA with Dunnet's post-test *p<0.05 vs unstimulated, n = 5. Image at top of graph is a representative composite western blot.

Figure 8. LPS does not induce fibronectin deposition (A) or IL-8 release (B).

Human lung fibroblasts were stimulated for 72 µg/ml lipopolysaccharide (LPS) prior to analysis of fibronectin deposition by ECM ELISA (A) or IL-8 release by ELISA (B). The profibrotic cytokine TGF-β₁ (1 ng/ml) was used as a positive control for fibronectin deposition. Data expressed as % of unstimulated (A) or pg/ml (B). Bars represent mean ± SEM. Data analysed by one-way ANOVA with Dunnet's post-test.*p<0.05 vs unstimulated, n = 5.