Exposure of Differentiated Airway Epithelial Cells to Volatile Smoke in vitro

Abstract

Background: Cigarette smoke (CS) is the predominant pathogenetic factor in the development of chronic bronchitis and chronic obstructive pulmonary disease. The knowledge about the cellular and molecular mechanisms underlying the smoke-induced inflammation in epithelial cells is limited. Objectives: The aim of this study was to develop an in vitro model to monitor the effects of volatile CS on differentiated airway epithelial cells. Methods: The airway epithelial cell line MM-39 and primary human bronchial epithelial cells were cultivated as air-liquid interface cultures and exposed directly to volatile CS. We used two types of exposure models, one using ambient air, the other using humidified and warm air. Cytokine levels were measured by quantitative PCR and ELISA. Phosphorylation of p38 MAP kinase was assessed by Western blot analysis. To reduce the smoke-induced inflammation, antisense oligonucleotides directed against the p65 subunit of NF-ĸB were applied. Results: Exposure of epithelia to cold and dry air resulted in a significant inflammatory response. In contrast, exposure to humidified warm air did not elicit a cellular response. Stimulation with CS resulted in upregulation of mRNA for IL-6 and IL-8 and protein release. Exposure to CS combined with heat-inactivated bacteria synergistically increased levels of the cytokines. Reactions of differentiated epithelial cells to smoke are mediated by the MAP kinase p38 and the transcription factor NF-ĸB. Conclusions: We developed an exposure model to examine the consequences of direct exposure of differentiated airway epithelial cells to volatile CS. The model enables to measure the cellular reactions to smoke exposure and to determine the outcome of therapeutic interventions.

Neutrophil Elastase Differentially Regulates Interleukin 8 (IL-8) and Vascular Endothelial Growth Factor (VEGF) Production by Cigarette Smoke Extract

“…CS contains more than 4,000 chemicals and additives (22), many of which are volatile. Although in vitro treatment with CSE may not accurately reflect in vivo exposure to CS, reports show that when airway epithelial cells were exposed to volatile CS IL-8 was released into the culture medium via p38 (23). It is well known that neutrophils recruited by the action of IL-8 release NE.…”

Suppression of pulmonary innate host defense in smokers

“…Tissue cultures were exposed to volatile cigarette smoke (CS) as described previously 17 . Briefly, tissue cultures were exposed to CS for 15 min (= 3 cigarettes).…”

“…RNA isolation, reverse transcription, and quantitative PCR were performed as described elsewhere 17 . GAPDH primer (sense, 5'-GAAGGTGAAGGTCGGAGTC-3'; antisense, 5'-GAAGATGGTGATGGGATTTC-3'), hBD-1 primer (sense, 5'-GCCTCAGGTGGTAACTTTCTCA -3'; antisense 5'-GCGTCATTTCTTCTGGTCACT -3'), and hBD-2 primer (sense 5'-TCAGCTCCTGGTGAAGCTC -3'; antisense 5'-GGGCAAAAGACTGGATGACA -3') were purchased from TIB Molbiol (Berlin, Germany).…”

“…Smoke is generally thought to affect proinflammatory pathways including MAP kinases p38, ERK1/2, and JNK or NF-κB 17 . Acrolein is a major product of organic combustions and inhibits NF-κB-activation by interaction with IkappaB kinase (IKK) 29 .…”

Role of CXCR2 in cigarette smoke-induced lung inflammation

“…The importance of these chemokines in promoting inflammation has been investigated in vitro and in vivo. Bronchoalveolar lavage (BAL) fluid from COPD patients contains increased neutrophils, TNF-␣, and IL-8 (25,29), whereas we and others have shown that cigarette smoke extract stimulates human lung cells (epithelial cells, fibroblasts, and macrophages) to release IL-8 (3,19,20,30). In mice, MIP-2 is essential for neutrophil recruitment to the lungs following inflammatory injury (22).…”