The chronicity of bronchial asthma is attributed to persistent airway inflammation and to a variety of structural changes, or remodelling, that includes smooth muscle and goblet cell hyperplasia.To investigate the mechanisms of airway remodelling, the current authors used an established allergen (ovalbumin; OVA)-driven rodent model (the Brown Norway rat).Brown Norway rats were sensitised to OVA and challenged three times at 5-day intervals to evoke airway remodelling. The effects of an epidermal growth factor (EGF) receptor inhibitor, AG1478, and a cysteinyl leukotriene-1 receptor antagonist, montelukast, on epithelial and airway smooth muscle (ASM) cell proliferation in vivo in response to repeated OVA challenge were tested. Three challenges with leukotriene (LT)D 4 were given, to examine their effects on remodelling with and without AG1478 pretreatment.OVA challenges caused ASM hyperplasia, with an increase in mass, epithelial cell proliferation and goblet cell proliferation. AG1478 prevented the changes, as did montelukast. Multiple OVA challenges increased heparin-binding EGF-like growth factor but not EGF expression by airway epithelium. LTD 4 reproduced the changes in remodelling induced by OVA and this was blocked by AG1478.Allergen-induced airway epithelial and airway smooth muscle remodelling is mediated by cysteinyl leukotrienes via the cysteinyl leukotriene-1 receptor with downstream effects on the epidermal growth factor receptor axis.KEYWORDS: Allergy, inflammation, lung, signal transduction A sthmatic airways often show extensive and complex remodelling [1][2][3][4]. The growth of smooth muscle has the potential to have the most significant pathophysiological consequences through excessive airway narrowing and airway hyperresponsiveness [5,6]. Increase in airway smooth muscle (ASM) in the airways has been associated with the severity of asthma [3,7], and when present in excess in the large airways is associated with mortality [8]. It is known that hyperplasia of smooth muscle in animal models [9][10][11] and both hyperplasia and hypertrophy in airway specimens from human subjects [12,13] contribute to the increase in ASM mass. It has also been proposed that migration of subepithelial myofibroblasts may add to the tissue mass [7].The mechanism of the growth response of muscle is quite uncertain, although several descriptive studies of growth factor expression in human airway tissues have been reported [14][15][16] and many growth factors have been demonstrated to have mitogenic effects on ASM in culture [17][18][19][20]. Cysteinyl (cys)-leukotrienes (LTs) are known to be involved in allergen-induced ASM cell proliferation in vivo [21,22] but in vitro these substances are weak mitogens for ASM [23,24]. In the sensitised mouse, cys-LT 1 receptor (cys-LT 1 R) antagonism prevents an increase in ASM thickening after repeated allergen challenge [25,26]. It is possible that the effects of cys-LTs in vivo are indirect and mediated by altering the expression of or potentiating the effects of tyrosi...

Taken together, our results indicate great potential for the use of S28463 as an antiinflammatory therapeutic agent for the management of chronic asthma.

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Patients with cystic fibrosis (CF) suffer from asthma-like symptoms and gastrointestinal cramps, attributed to a mutation in the CF transmembrane conductance regulator (CFTR) gene present in a variety of cells. Pulmonary manifestations of the disease include the production of thickened mucus and symptoms of asthma, such as cough and wheezing. A possible alteration in airway smooth muscle (ASM) cell function of patients with CF has not been investigated. The aim of this study was to determine whether the (CFTR) channel is present and affects function of human ASM cells. Cell cultures were obtained from the main or lobar bronchi of patients with and without CF, and the presence of the CFTR channel detected by immunofluorescence. Cytosolic Ca(2+) was measured using Fura-2 and dual-wavelength microfluorimetry. The results show that CFTR is expressed in airway bronchial tissue and in cultured ASM cells. Peak Ca(2+) release in response to histamine was significantly decreased in CF cells compared with non-CF ASM cells (357 +/- 53 nM versus 558 +/- 20 nM; P < 0.001). The CFTR pharmacological blockers, glibenclamide and N-phenyl anthranilic acid, significantly reduced histamine-induced Ca(2+) release in non-CF cells, and similar results were obtained when CFTR expression was varied using antisense oligonucleotides. In conclusion, these data show that the CFTR channel is present in ASM cells, and that it modulates the release of Ca(2+) in response to contractile agents. In patients with CF, a dysfunctional CFTR channel could contribute to the asthma diathesis and gastrointestinal problems experienced by these patients.

The effects of remodeling of airway smooth muscle (SM) by hyperplasia on airway SM contractility in vivo are poorly explored. The aim of this study was to investigate the relationship between allergen-induced airway SM hyperplasia and its contractile phenotype. Brown Norway rats were sensitized with ovalbumin (OVA) or saline on day 0 and then either OVA-challenged once on day 14 and killed 24 h later or OVA-challenged 3 times (on days 14, 19, and 24) and killed 2 or 7 days later. Changes in SM mass, expression of total myosin, SM myosin heavy chain fast isoform (SM-B) and myosin light chain kinase (MLCK), tracheal contractions ex vivo, and airway responsiveness to methacholine (MCh) in vivo were assessed. One day after a single OVA challenge, the number of SM cells positive for PCNA was greater than for control animals, whereas the SM mass, contractile phenotype, and tracheal contractility were unchanged. Two days after three challenges, SM mass and PCNA immunoreactive cells were increased (3-and 10-fold, respectively; P Ͻ 0.05), but airway responsiveness to MCh was unaffected. Lower expression in total myosin, SM-B, and MLCK was observed at the mRNA level (P Ͻ 0.05), and total myosin and MLCK expression were lower at the protein level (P Ͻ 0.05) after normalization for SM mass. Normalized tracheal SM force generation was also significantly lower 2 days after repeated challenges (P Ͻ 0.05). Seven days after repeated challenges, features of remodeling were restored toward control levels. Allergen-induced hyperplasia of SM cells was associated with a loss of contractile phenotype, which was offset by the increase in mass. phenotype; animal model; myosin heavy chain isoform STRUCTURAL AIRWAY REMODELING has been widely reported in both human asthma (4, 13) and animal models of experimental asthma (11,23,37). Most tissues of the airway wall are affected by remodeling, and the observed changes include shedding of the epithelium, thickening of the reticular basement membrane, increased mucus gland size and number, goblet cell differentiation, angiogenesis, and augmentation of the airway smooth muscle (SM) mass (4). The changes in airway wall tissues are likely to be of clinical importance since they may control airway caliber, airway elasticity, and responsiveness and may cause symptoms related to mucus overproduction (4). Airway SM remodeling is a reported feature of several animal models of allergic asthma (11,12,22), including the Brown Norway (BN) rat (19,26,31). The increase in SM mass, observed in asthma of varying severity (5, 29, 36), has been proposed to be sufficient to account for altered airway responsiveness (16). The augmentation of airway SM mass has been attributed to hyperplasia through evidence of cell proliferation quantified by bromodeoxyuridine incorporation or PCNA expression in vivo (11,19,28,37). Susceptibility to SM remodeling may be a risk factor for asthma; cultured airway SM cells from asthmatic subjects were shown to have a higher proliferation rate than cells from normal subjects (14), sim...

Airway epithelial cells release proinflammatory mediators that may contribute to airway remodeling and leukocyte recruitment. We explored the hypothesis that leukotriene D₄ (LTD₄) may trigger the release of proremodeling factors through activation of the EGF receptor (EGFR). We particularly focused on the effects of LTD₄ on release of heparin-binding EGF-like factor (HB-EGF) and IL-8 (CXCL8), a potent neutrophil chemoattractant that may be released downstream of EGFR activation. To address this hypothesis, both primary (NHBE) and transformed bronchial human epithelial cells (BEAS-2B) were grown on an air-liquid interface and stimulated with LTD₄. HB-EGF and CXCL8 were evaluated by ELISA in cell culture supernatants. To explore the EGFR signaling pathway, we used a broad-spectrum matrix metalloproteinase (MMP) inhibitor, GM-6001, two selective EGFR tyrosine kinase inhibitors, AG-1478 and PD-153035, an HB-EGF neutralizing antibody, and a specific small interfering RNA (siRNA) against the EGFR. Expression of the CysLT₁ cysteinyl leukotriene receptor was demonstrated by RT-PCR and immunocytochemistry in both BEAS-2B and NHBE cells. Four hours after stimulation with LTD₄, HB-EGF and CXCL8 were significantly increased in cell culture supernatant. GM-6001 and montelukast, a specific CysLT₁ receptor antagonist, blocked the LTD₄-induced increase in HB-EGF. All inhibitors/antagonists decreased LTD₄-induced CXCL8 release. siRNA against EGFR abrogated CXCL8 release following stimulation with LTD₄ and exogenous HB-EGF. These findings suggest LTD₄ induced EGFR transactivation through the release of HB-EGF in human bronchial epithelial cells with downstream release of CXCL8. These effects may contribute to epithelial-mediated airway remodeling in asthma and other conditions associated with cysteinyl leukotriene release.

Sphingosine-1-phosphate (S1P) is an immunomodulatory lipid mediator that plays an important role in lymphocyte trafficking. Elevated levels of S1P are found in bronchoalveolar lavage (BAL) fluid of patients with asthma; however, its role in disease is not known. FTY720, a synthetic analog of S1P, has been shown to abrogate allergic inflammation and airway hyperresponsiveness following acute allergen challenge. However, its effects on asthmatic airway remodeling induced by repeated allergen exposure are unknown. Ovalbumin (OVA)-sensitized rats were challenged on days 14, 19, and 24 after sensitization. FTY720 or vehicle (PBS) therapy was administered 1 h prior to each challenge. BAL fluid and quantitative histological analysis were performed 48 h after the last challenge. FTY720 inhibited OVA-induced features of airway remodeling including increased airway smooth muscle mass and bronchial neovascularization, without affecting lymphocyte numbers in secondary lymphoid organs. Furthermore, CD3+ cells adjacent to airway smooth muscle bundles were increased in OVA-challenged rats but the increase was inhibited by FTY720. There was an expansion of bronchus-associated lymphoid tissue following FTY720 treatment of OVA-challenged animals. Real-time quantitative PCR revealed that Th2-associated transcription factors were inhibited following FTY720 therapy. Airway remodeling is a cardinal feature of severe asthma. These results demonstrate that allergen-driven airway remodeling can be inhibited by FTY720, offering potential new therapies for the treatment of severe asthma.

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