Abstract
BackgroundFibroblasts are implicated in sub-epithelial fibrosis in remodeled asthmatic airways and contribute to airway inflammation by releasing cytokines and other mediators. Fibroblast activity is influenced by members of the leukotriene family of bronchoconstrictor and inflammatory mediators, but it is not known whether human bronchial fibroblasts can synthesize leukotrienes.MethodsThe expression of leukotriene biosynthetic enzymes and receptors was investigated in primary fibroblasts from the bronchi of normal and asthmatic adult subjects using RT-PCR, Western blotting, immunocytochemistry and flow cytometry.ResultsThese techniques revealed that human bronchial fibroblasts from both subject groups constitutively express 5-lipoxygenase, its activating protein FLAP, the terminal enzymes leukotriene A4 hydrolase and leukotriene C4 synthase, and receptors for leukotriene B4 (BLT1) and cysteinyl-leukotrienes (CysLT1). Human bronchial fibroblasts generated immunoreactive leukotriene B4 and cysteinyl-leukotrienes spontaneously and in increased amounts after calcium-dependent activation. Flow cytometry showed that human bronchial fibroblasts transformed to a myofibroblast-like phenotype by culture with transforming growth factor-β1 expressed 320–400% more immunofluorescence for leukotriene C4 synthase and CysLT1 receptors, with 60–80% reductions in leukotriene A4 hydrolase and BLT1 receptors.ConclusionThese results indicate that human bronchial fibroblasts may not only respond to exogenous leukotrienes but also generate leukotrienes implicated in narrowing, inflammation and remodeling of the asthmatic airway.
Highlights
Fibroblasts are implicated in sub-epithelial fibrosis in remodeled asthmatic airways and contribute to airway inflammation by releasing cytokines and other mediators
Basal expression of LT pathway enzymes in human bronchial fibroblasts To examine whether HBFs constitutively express leukotriene pathway enzymes, baseline levels of 5-LO, FLAP, LTA4 hydrolase and LTC4 synthase mRNA and protein were assessed by RT-PCR, SDS-PAGE/Western immunoblotting, immunocytochemistry and flow cytometry
SDS-PAGE/Western immunoblotting (Figure 1B) revealed that fibroblasts constitutively expressed 5-LO, FLAP, LTA4 hydrolase and LTC4 synthase proteins, with immunopositive bands corresponding to the expected molecular weights of approximately 80 kDa, 18 kDa, 70 kDa and 18 kDa respectively. 5-LO and FLAP in fibroblasts comigrated with recombinant 5-LO and FLAP standards, and the intensities of immunostaining for all four enzymes were similar to those observed in equal amounts of total cellular protein extracted from peripheral blood leukocytes (PBL) (Figure 1B)
Summary
Fibroblasts are implicated in sub-epithelial fibrosis in remodeled asthmatic airways and contribute to airway inflammation by releasing cytokines and other mediators. Airway inflammation and remodeling are prominent features of the asthmatic lung, characterized by leukocyte infiltration, oedema and mucus hypersecretion, combined with sub-epithelial fibrosis and smooth muscle hypertrophy. Fibroblasts are the most abundant cell type of the lung interstitium and play a significant role in airway inflammation by expressing an array of cytokines and adhesion molecules which play key roles in infiltration and activation of eosinophils and other leukocytes [1]. Members of the cysteinylleukotriene sub-family increase the proliferation of bronchial epithelial cells and airway smooth muscle cells [6,7]. In a murine model of chronic allergic asthma with fibrosis, blockade of cysteinyl-leukotriene activity by montelukast significantly reduced eosinophil infiltration, mucus plugging, and smooth muscle hyperplasia, and completely prevented the development of sub-epithelial fibrosis [10]. We postulate that human bronchial fibroblasts (HBFs) themselves are capable of generating leukotrienes that may influence fibrosis and other remodeling processes by autocrine or paracrine mechanisms
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