Search

Chronic mucus hypersecretion (CMH) contributes to the morbidity and mortality of asthma, and remains uncontrolled by current therapies in the subset of patients with severe, steroid-resistant disease. Altered cross-talk between airway epithelium and airway smooth muscle cells (ASMCs), driven by pro-inflammatory cytokines such as interleukin (IL)-1β, provides a potential mechanism that influences CMH. This study investigated mechanisms underlying CMH by comparing IL-1β-induced gene expression profiles between asthma and control-derived ASMCs and the subsequent paracrine influence on airway epithelial mucus production in vitro IL-1β-treated ASMCs from asthmatic patients and healthy donors were profiled using microarray analysis and ELISA. Air-liquid interface (ALI)-cultured CALU-3 and primary airway epithelial cells were treated with identified candidates and mucus production assessed.The IL-1β-induced CCL20 expression and protein release was increased in ASMCs from moderate compared with mild asthmatic patients and healthy controls . IL-1β induced lower MIR146A expression in asthma-derived ASMCs compared with controls. Decreased MIR146A expression was validated in vivo in bronchial biopsies from 16 asthmatic patients versus 39 healthy donors. miR-146a-5p overexpression abrogated CCL20 release in ASMCs. CCL20 treatment of ALI-cultured CALU-3 and primary airway epithelial cells induced mucus production, while CCL20 levels in sputum were associated with increased levels of CMH in asthmatic patients.Elevated CCL20 production by ASMCs, possibly resulting from dysregulated expression of the anti-inflammatory miR-146a-5p, may contribute to enhanced mucus production in asthma., Competing Interests: Conflict of interest: M. Van den Berge reports research grants (paid to university) from Teva, Chiesi and GlaxoSmithKline, outside the submitted work. J.L. Black reports grants from the National Health and Medical Research Council, during the conduct of the study. J.K. Burgess reports grants from the National Health and Medical Research Council, during the conduct of the study., (Copyright ©ERS 2018.)

We hypothesised that the response to cigarette smoke in airway smooth muscle (ASM) cells from smokers with chronic obstructive pulmonary disease (COPD) would be intrinsically different from smokers without COPD, producing greater pro-inflammatory mediators and factors relating to airway remodelling. ASM cells were obtained from smokers with or without COPD, and then stimulated with cigarette smoke extract (CSE) or transforming growth factor-β1. The production of chemokines and matrix metalloproteinases (MMPs) were measured by ELISA, and the deposition of collagens by extracellular matrix ELISA. The effects of CSE on cell attachment and wound healing were measured by toluidine blue attachment and cell tracker green wound healing assays. CSE increased the release of CXCL8 and CXCL1 from human ASM cells, and cells from smokers with COPD produced more CSE-induced CXCL1. The production of MMP-1, -3 and -10, and the deposition of collagen VIII alpha 1 (COL8A1) were increased by CSE, especially in the COPD group which had higher production of MMP-1 and deposition of COL8A1. CSE decreased ASM cell attachment and wound healing in the COPD group only. ASM cells from smokers with COPD were more sensitive to CSE stimulation, which may explain, in part, why some smokers develop COPD., (©ERS 2014.)

Glycosaminoglycans (GAG) are essential extracellular matrix molecules which regulate tissue flexibility, a parameter that is reduced in airways of patients with asthma and chronic obstructive pulmonary disease (COPD). We investigated the expression of GAG and their metabolising enzymes in primary human airway smooth muscle cells (ASMC) obtained from healthy donors (controls) and patients with asthma or COPD. Total GAG synthesis was assessed by [(3)H]-glucosamine incorporation. GAG were isolated, purified, fractionated by electrophoresis and characterised using specific GAG-degrading enzymes. Secretion of hyaluronic acid (HA) by ASMC from patients with asthma or COPD was significantly decreased compared with controls. RT-PCR analysis and western blotting revealed that this decrease was associated with a significant reduction in the expression of HA synthase-1 and -2 and a significant increase of hyaluronidase-1. Furthermore, the expression of the HA receptor CD44 was significantly decreased, whereas the receptor for HA-mediated motility was not expressed in asthma or COPD. Our results indicate that there is a decreased expression of HA in asthma and COPD associated with a synergistic regulation of HA metabolising enzymes that may regulate the pathological airway remodelling in these lung diseases.

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not "cure" asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

Lymphangioleiomyomatosis (LAM) is associated with abnormal airway smooth muscle that leads to the characteristic pathology of lung nodule formation and destruction of lung tissue. The current authors have previously identified abnormal behaviour of airway smooth muscle cells from patients with asthma. In this study, cells and tissue sections derived from patients with LAM (n=7), asthma (n=8), and nonasthmatic controls (n=9) were compared. The presence of the antigen human melanosome (HM)B-45 was investigated, along with the proliferation and release of extracellular matrix proteins, release of endogenous prostaglandin E2 (PGE2), vascular endothelial growth factor and connective tissue growth factor, and the expression of integrins. Positive HMB-45 staining was found in all LAM patients and no controls. Proliferation of LAM cells was not different from control cells nor was its inhibition by beta-agonists, corticosteroids, rapamycin or PGE2. However, endogenous PGE2 levels were markedly decreased in LAM cells, and this was associated with decreased expression of the inducible form of cyclooxygenase (COX-2). The increased levels of connective tissue growth factor seen in asthma cells were not observed in LAM. Elastin mRNA in response to transforming growth factor-beta stimulation was markedly lower in LAM cells than either asthma or control cells. In conclusion, lymphangioleiomyomatosis cells exhibit abnormal properties in vitro that may contribute to pathophysiology and symptomatology in patients with lymphangioleiomyomatosis.

Breathing at volumes lower than functional residual capacity (FRC) can induce changes in nonasthmatic airways consistent with the behaviour of asthmatic airways. This study investigated the chronic effect of breathing at volumes lower than FRC on the contractility of airway smooth muscle and myosin light chain kinase (MLCK) content and activity. Sheep of three age groups (neonate, adolescent and adult) had their FRC reduced by approximately 25%, for 4 weeks using a leather corset. Contractile responses to carbachol were then recorded in isolated tracheal strips and bronchial rings. MLCK content and activity were assessed by immunoblotting. The rate of stress generation increased in the bronchial smooth muscle of both adult and adolescent but not neonatal corseted sheep: adolescent corseted versus control, 65.0 +/- 4.1 versus 103.4 +/- 7.0 s (to reach 50% maximum stress), respectively; and adult corseted versus control, 57.0 +/- 6.4 versus 93.4 +/- 8.2 s, respectively. This was not due to increases in either bronchial or tracheal smooth muscle amount or MLCK content and activity. The present results indicate that chronic breathing at low lung volumes increases the rate of stress generation in airway smooth muscle.

In vivo agonist delivery to the lungs is characterized by absorption through the mucosal layer followed by access to the smooth muscle. When agonists are applied to perfused bronchial segments, a difference in potency to agonists applied to the serosal (outside) or mucosal (inside) surface can be demonstrated. In order to elucidate this effect in canine bronchial segments, we adapted a method to assess responsiveness of agonists applied to the inside or outside surface of canine bronchial segments. A 2 cm "fluid-tight" length of bronchus was cannulated and mounted in a perfusion chamber. Auxotonic contraction of the bronchus displaced fluid inside the segment up a column and the change in height of fluid within the column (afterload) was measured as a change in hydrostatic pressure (volumetric). We assessed the optimal conditions for measuring bronchial responsiveness to acetylcholine. Neither stretching segments lengthways from 100 to 140% of resting length, nor altering the transmural pressure from 3 to 21 cmH2O had a significant effect on the potency of acetylcholine applied to the outside surface. Both acetylcholine (n=7) and methacholine (n=4) were approximately 10-fold more potent when applied to the outside surface than the inside surface of bronchial segments (p<0.0001). Furthermore, mechanical removal of the epithelium (n=6) led to a 67-fold increase in potency of acetylcholine applied to the inside surface compared with segments with epithelium. The advantages that this system has over previously reported methods include: 1) agonists can be injected at a low flow rate so as to minimize epithelial stress; 2) changes in luminal volume are measured under conditions of no flow; 3) after-load can be varied such that contraction is either practically isotonic or auxotonic; and 4) a "barrier effect" of the epithelium of canine bronchial segments can be demonstrated. This new adapted method will provide us with the means to assess the relationship between in vitro and in vivo responsiveness in human bronchi.

The mast cell plays a pivotal role in the early asthmatic response via release of mediators, which directly influence airway smooth muscle tone. Canine mast cell tryptase has been reported to potentiate the contractile response of canine isolated airways to histamine. The aim of this study was to investigate whether human mast cell tryptase potentiated contractile responses in human isolated bronchi. The effect of tryptase differed according to the sensitization status of the bronchi. In lung tissue from sensitized patients (those whose bronchial tissue contracted in response to the application of any of four common antigens) 90 ng.mL-1 of human purified lung tryptase markedly potentiated the contractile response to histamine. The maximal response as a percentage of maximal contraction to acetylcholine was 80 +/- 8% in control tissues and 119 +/- 6% in tryptase treated tissues (n = 4; p < 0.05). Tryptase, at a dose of 200 ng.mL-1, also potentiated responses but to a lesser degree, 100 +/- 5% (n = 4; p < 0.05). In nonsensitized bronchi, neither 90 nor 200 ng.mL-1 tryptase had any significant effect on histamine responses. The increased response in the presence of tryptase in sensitized tissue was inhibited by the calcium voltage-dependent channel antagonist, verapamil (10(-6) M). We have shown, for the first time, that human mast cell tryptase potentiates contraction in sensitized bronchi via a calcium-related mechanism. These findings provide a link between a mast cell derived product and in vitro human airway hyperresponsiveness.

In recent years, platelet-activating factor (PAF) has been strongly implicated as a mediator involved in asthma. In non-asthmatic subjects, aerosolized PAF has been shown to cause bronchoconstriction. The mechanism of this in vivo effect is unknown. We have previously shown that PAF causes a contraction of human isolated bronchus that varies in magnitude between patients, and within tissues from the same patient. To examine the possibility that this variability in contraction was secondary to PAF-induced release of mediators from inflammatory or epithelial cells within the tissue, we examined the relationship between contractile responses to PAF and the presence of inflammatory or epithelial cells. We studied eight tissues from five patients. Of the eight tissues, four contracted, whilst four failed to contract, to PAF (7 x 10(-7) M). After the contractile response to PAF had been assessed by observing changes in isometric tone in vitro, bronchial rings were examined histologically to enable the quantification of inflammatory cell numbers and intact epithelium. No significant correlation was observed between the magnitude of contractions and numbers of eosinophils, neutrophils, lymphocytes, plasma cells, total cells or percentage intact epithelium. We conclude that it is unlikely that the variability in response to PAF in human isolated airways is related to the variability in inflammatory cell numbers or to the presence of epithelium. Thus, the contraction induced by PAF is probably not mediated via the release of a secondary mediator from the particular cells examined in this study.

This study examines the possibility that products of neutrophils and eosinophils could increase the responsiveness of human isolated bronchial tissue. Neutrophils and eosinophils were isolated from the peripheral blood of healthy volunteers. The cells were incubated with 1 microM calcium ionophore A23187 for 10-15 min then centrifuged, the supernatant collected and stored at -70 degrees C. Human bronchial rings (2-3 mm diameter, 3-4 mm long) were prepared from specimens resected at thoracotomy. The tissues were suspended in organ baths under a 1 g load and changes in tension measured isometrically. Stable contractions to bolus doses of histamine (0.1-10 microM) or to electrical field stimulation (40-100 V, 4-16 Hz, 1 ms for 20 s) were established. Supernatant from 106 neutrophils or 105 eosinophils was then added and tissue responsiveness reassessed. Neutrophil supernatant increased tissue responsiveness to histamine and electrical field stimulation by 54 +/- 17% (n = 5, p less than 0.05) and 18 +/- 7% (n = 6, p less than 0.05), respectively. Eosinophil supernatant increased the histamine response by 60 +/- 23% (n = 8, p less than 0.05) while tissue responsiveness to electrical field stimulation was unchanged (n = 3). Thus, as neutrophils and eosinophils can change the responsiveness of human bronchus in vitro it is possible that they do this in vivo and may not simply be temporally related to the development of bronchial hyperresponsiveness.

This study investigated the direct and indirect effects of platelet activating factor (PAF, PAF-acether) on human isolated airways. PAF caused a single non-repeatable contraction which was found to be variable both between lung samples and within tissues from the same lung. The range of contractions induced by PAF, 7 X 10(-8), M and 7 X 10(-7) M was 2-55% and 3-72% of the maximal response to carbachol, respectively. The PAF antagonist WEB 2086, 10(-6) M, inhibited the contraction induced by PAF, 7 X 10(-7) M, (p less than 0.05, n = 5). PAF caused a significant potentiation of the contractile effects of bolus doses of histamine. This potentiation was inhibited by WEB 2086, 10(-6) M, (p less than 0.05, n = 5). These results suggest firstly that the bronchoconstriction observed in man after PAF administration could be the result of an action of PAF on the smooth muscle or result from the action of a second mediator released by PAF from cells within the lung. Secondly, the bronchial hyperresponsiveness observed after PAF administration could be due to a specific alteration in smooth muscle sensitivity. The fact that WEB 2086 inhibited the contractile response to PAF and the potentiation of histamine-induced contraction would suggest that PAF receptors mediate these responses.