Endotoxin and pro‐inflammatory cytokines stimulate endothelin‐I expression and release by airway epithelial cells

Abstract

Endothelin is a potent bronchoconstrictor peptide first identified as a novel vasoconstrictor produced by vascular endothelial cells. Recent reports suggest that airway epithelial cells are also capable of releasing this active peptide. To investigate the regulatory mechanism of endothelin expression, we studied the effects of endotoxin and pro-inflammatory cytokines such as interleukin-1 and tumour necrosis factor on the expression and release of endothelin-1 by airway epithelial cells. Both endotoxin and the cytokines stimulated endothelin-1 release by human bronchial epithelial cells. Northern blot analysis showed increased expression of preproendothelin-1 mRNA by these factors. These results suggested that airway epithelial cells might play a role in the local airway smooth muscle tone through the production of endothelin, which might be upregulated by inflammatory products in the airways.

Erythromycin and clarithromycin attenuate cytokine-induced endothelin-1 expression in human bronchial epithelial cells

“…We used primary and secondary passaged cells for the experiments. In all preparations of primary and secondary passaged bronchial epithelial cells, no less than 98% of the cells were positive to keratin, but negative to vimentin, indicating that the cells were of epithelial cell origin as described [8,18].…”

“…The specific immunoreactivity for ET-1 in culture supernatants was measured by sandwich enzyme-linked immunosorbent assay (ELISA) kits previously reported by SUZUKI et al [8,21]. Each sample was assayed in duplicate.…”

“…Normal human bronchial epithelial cells were prepared by the method reported previously [8,16,17]. Briefly, a piece of macroscopically and microscopically normal human lobe or segmental bronchus (about 7 mm in width) was obtained either at the time of lung tumour resection or on autopsy.…”

“…The medium was changed on day 1 and then every 2 days. Confluent monolayers of epithelial cells were stained with anti-keratin (KL-1, Immunotech, Marseille, France) or anti-vimentin (DAKO-Vimentin, DAKOPatts, Glostrup, Denmark), or with control immunoglobulin-G1 (IgG1) monoclonal antibodies using the avidin-biotin complex method [8,18]. We used primary and secondary passaged cells for the experiments.…”

“…The cells were placed on to collagen-coated 24-well flat-bottom tissue culture plates (Koken, Tokyo, Japan) at a density of 5×10 4 cells·well -1 in hormonally defined Ham's F12 medium (HD-F12) as reported previously [8,16,17]. HD-F12 contained 1% penicillin-streptomycin, 5 µg·mL -1 insulin (GIBCO), 5 µg·mL -1 transferrin (GIBCO), 25 ng· mL -1 epidermal growth factor (Collaborative Research Corp., Lexington, MA), 15 µg·mL -1 endothelial cell growth supplement (Collaborative Research Corp.), 2×10 -10 M triiodothyronin (GIBCO), and 10 -7 M hydrocortisone (GIBCO).…”