Inflammatory Profile Of Human Vocal Fold Fibroblasts In Response To Cigarette Smoke Extract

Abstract

Introduction: Tobacco smoke is the dominant risk for laryngeal squamous neoplasia and chronic laryngitis. The hallmark of many such laryngeal diseases is the onset of mucosal inflammation, which can affect the critical functions of swallowing, breathing, and voice. While we currently have a limited understanding of how mucosal inflammation is manifested in response to cigarette smoke, we know that tissue specific fibroblasts are able to mediate chronic inflammation of other tissues through the secretion of soluble cytokines and paracrine regulation. Ultimately these processes can influence the outcomes of diseases such as cancer. In Reinke's edema of the vocal fold, lamina propria fibroblasts are the primary cell type exposed to the inflammation induced edema associated with cigarette smoke exposure. Here we profile the cytokine response of human vocal fold fibroblasts to cigarette smoke exposure in vitro. Methods: Cigarette smoke extract (CSE) was prepared in accord with investigators studying the effects of cigarette smoke in vitro. One Marlboro cigarette with a removed filter was combusted into a 30 mL syringe at a rate of 3 extractions/minute over 5 minutes. The smoke was bubbled into 10 mL of cell culture media. This CSE was neutralized to pH 7.4, filtered, adjusted to OD 1.0 (320nm) with a spectrophotometer and denoted 10% CSE. The control for CSE was air bubbled into the media. TNF-α (10 ng/mL) was used as a proinflammatory cytokine control. Immortalized human vocal fold fibroblasts (hVFF) were transiently treated for 3 hours with 0.5 and 1% CSE before being washed 2 times with 1x PBS and then returned to normal media for 24 hours. Trypan blue exclusion determined that cell death did not occur with CSE treatment. Cytokine levels of the media were assessed by the Bio-Rad Bio-Plex 200 assay reader. Results: CSE (1.0%) had a significant increase in VEGF levels compared to control, CSE (0.5%) and TNF-α (TABLE). TNF-α treatment showed a significant increase in IL-8 and cytokine TNF-α levels compared to control and CSE exposure. Although CSE (1.0%) exposure did show the trend of increasing IL-8 levels, this increase was not statistically significant. Additionally, IL-1B, IL-12 and Eotoxin levels did not change. Conclusions: Transient exposure of 1/10 of a combusted cigarette (CSE, 1.0%) showed the specific secretion of the angiogenesis regulatory cytokine VEGF relative to other treatments. Therefore, hVFFs could participate in the angiogenesis necessary for inflammation through the paracrine regulation of other tissues in the larynx. Lastly, since fibroblasts can mediate the inflammation associated with certain diseases, altering the immunomodulatory response of hVFFs could prevent diseases associated with mucosal inflammation of the larynx.