Aging enhances susceptibility to cigarette smoke-induced inflammation through bronchiolar chemokines.

Cigarette smoking and aging are major risk factors for chronic obstructive pulmonary disease. An unsolved question is whether elderly lungs are particularly vulnerable to cigarette smoke (CS) exposure. In this study, we used a mouse model to test the hypothesis that aging increases the susceptibility to CS-induced pulmonary inflammation. We subjected 9-week-old and 69-week-old C57BL/6J mice to CS (whole-body exposure, 90 min/d), and evaluated neutrophil infiltration in the lungs, the levels of keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2 in bronchoalveolar lavage fluid, and mRNA expression in bronchiolar epithelium retrieved by laser capture microdissection. The 69-week-old mice showed a greater number of neutrophils and higher levels of bronchiolar KC and MIP-2 expression than 9-week-old mice after 9 days of CS exposure. Furthermore, single CS exposure induced the rapid up-regulation of KC and MIP-2 in bronchiolar epithelium in both 9-week-old and 69-week-old mice, and the much higher levels in 69-week-old mice were associated with greater nuclear translocation of NF-kappaB. In contrast, no age-related differences were observed in the bronchiolar expression of NF-E2-related factor 2-regulated antioxidant and detoxification genes, heme oxygenase-1, reduced nicotinamide adenine dinucleotide phosphate quinone reductase 1, and glutamate-cysteine ligase, modifier unit, or antioxidant activity in bronchoalveolar lavage fluid, regardless of CS exposure. In summary, aging increases susceptibility to CS-induced inflammation in a mouse model, and robust mRNA up-regulation and nuclear translocation of NF-kappaB in bronchiolar epithelium may be involved.

Decreased airway expression of vascular endothelial growth factor in cigarette smoke-induced emphysema in mice and COPD patients.

Vascular endothelial growth factor (VEGF) signaling is crucial for lung structure maintenance. Although VEGF deficiency plays a role in the pathogenesis of emphysema in animals, little is known about VEGF expression levels and functions, as well as VEGF receptors, in airway epithelial cells, which are in direct contact with the environment. In this study, C57BL/6J mice were exposed to cigarette smoke (CS) for short (approximately 10 days) and long (4-24 wk) time periods, and bronchiolar expressions of VEGF and its receptors VEGFR-1 and VEGFR-2 were examined. The relationships between the expressions of VEGF, VEGFR-1, and VEGFR-2 and smoking histories and/or chronic obstructive pulmonary disease (COPD) were examined in humans. The mRNA levels were quantified in bronchiolar epithelium harvested by laser capture microdissection in both mouse and human lung tissues or in human bronchial epithelium harvested by bronchoscopic brushing. The VEGF protein level was assessed by immunohistochemistry or enzyme-linked immunosorbent assay. Repeated CS exposure downregulated bronchiolar expressions of VEGF and both VEGF receptors at various time points prior to the development of emphysema. In humans, bronchiolar VEGF was significantly decreased in smokers with COPD compared to lifelong nonsmokers, as well as to smokers without COPD; however, there was no difference in bronchiolar VEGF levels between lifelong nonsmokers and smokers without COPD. On the other hand, bronchiolar VEGFR-2 was downregulated in smokers with and without COPD compared to lifelong nonsmokers. These findings suggest the association of downregulation of bronchiolar VEGF and its receptors with cigarette smoking and COPD.

Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure.

BACKGROUND: Bronchioles are critical zones in cigarette smoke (CS)-induced lung inflammation. However, there have been few studies on the in vivo dynamics of cytokine gene expression in bronchiolar epithelial cells in response to CS. METHODS: We subjected C57BL/6J mice to CS (whole body exposure, 90 min/day) for various periods, and used laser capture microdissection to isolate bronchiolar epithelial cells for analysis of mRNA by quantitative reverse transcription-polymerase chain reaction. RESULTS: We detected enhanced expression of keratinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) by bronchial epithelial cells after 10 consecutive days of CS exposure. This was mirrored by increases in neutrophils and KC, MIP-2, TNF-alpha, and IL-1beta proteins in the bronchoalveolar lavage (BAL) fluid. The initial inhalation of CS resulted in rapid and robust upregulation of KC and MIP-2 with concomitant DNA oxidation within 1 hr, followed by a return to control values within 3 hrs. In contrast, after CS exposure for 10 days, this initial surge was not observed. As the CS exposure was extended to 4, 12, 18 and 24 weeks, the bronchiolar KC and MIP-2 expression and their levels in BAL fluid were relatively dampened compared to those at 10 days. However, neutrophils in BAL fluid continuously increased up to 24 weeks, suggesting that neutrophil accumulation as a result of long-term CS exposure became independent of KC and MIP-2. CONCLUSION: These findings indicate variable patterns of bronchiolar epithelial cytokine expression depending on the duration of CS exposure, and that complex mechanisms govern bronchiolar molecular dynamics in vivo.

Human airway trypsin-like protease induces amphiregulin release through a mechanism involving protease-activated receptor-2-mediated ERK activation and TNF alpha-converting enzyme activity in airway epithelial cells.

Human airway trypsin-like protease (HAT), a serine protease found in the sputum of patients with chronic airway diseases, is an agonist of protease-activated receptor-2 (PAR-2). Previous results have shown that HAT enhances the release of amphiregulin (AR); further, it causes MUC5AC gene expression through the AR-epidermal growth factor receptor pathway in the airway epithelial cell line NCI-H292. In this study, the mechanisms by which HAT-induced AR release can occur were investigated. HAT-induced AR gene expression was mediated by extracellular signal-regulated kinase (ERK) pathway, as pretreatment of cells with ERK pathway inhibitor eliminated the effect of HAT on AR mRNA. Both HAT and PAR-2 agonist peptide (PAR-2 AP) induced ERK phosphorylation; further, desensitization of PAR-2 with a brief exposure of cells to PAR-2 AP resulted in inhibition of HAT-induced ERK phosphorylation, suggesting that HAT activates ERK through PAR-2. Moreover, PAR-2 AP induced AR gene expression subsequent to protein production in the cellular fraction through the ERK pathway indicating that PAR-2-mediated activation of ERK is essential for HAT-induced AR production. However, in contrast to HAT, PAR-2 AP could not cause AR release into extracellular space; it appears that activation of PAR-2 is not sufficient for HAT-induced AR release. Finally, HAT-induced AR release was eliminated by blockade of tumour necrosis factor alpha-converting enzyme (TACE) by the TAPI-1 and RNA interference, suggesting that TACE activity is necessary for HAT-induced AR release. These observations show that HAT induces AR production through the PAR-2 mediated ERK pathway, and then causes AR release by a TACE-dependent mechanism.