Suzanne Zuyderduyn

Treating asthma means treating airway smooth muscle cells

Asthma is characterised by airway hyperresponsiveness, airway inflammation and airway remodelling. Airway smooth muscle cells are known to be the main effector cells of airway narrowing. In the present paper, studies will be discussed that have led to a novel view of the role of airway smooth muscle in the pathogenesis of asthma in which airway hyperresponsiveness, remodelling and inflammation are, at least in part, attributable to airway smooth muscle. Furthermore, how this new view may lead to a change in the phenotyping and treatment of patients with asthma will be discussed.

Enhanced pulmonary leptin expression in patients with severe COPD and asymptomatic smokers

Background: Chronic obstructive pulmonary disease (COPD) is characterised by an abnormal inflammatory reaction of the lungs involving activation of epithelial cells. Leptin is a pleiotropic cytokine important in the regulation of immune responses via its functional receptor Ob-Rb. This study was undertaken to test the hypothesis that severe COPD is associated with increased leptin expression in epithelial cells. Methods: Immunohistochemistry for leptin was performed on peripheral lung specimens from 20 patients with COPD (GOLD stage 4), 14 asymptomatic ex-smokers and 13 never smokers. Leptin and Ob-Rb mRNA expression were determined by rtPCR in cultured primary bronchial epithelial cells and primary type II pneumocytes. NCI-H292 and A549 cell lines were used to study functional activation of leptin signalling. Results: Leptin immunoreactivity in lung tissue was observed in bronchial epithelial cells, type II pneumocytes, macrophages (tissue/alveolar) and interstitial lymphocytic infiltrates. rtPCR analysis confirmed pulmonary leptin and Ob-Rb mRNA expression in primary bronchial epithelial cells and pneumocytes. Leptin-expressing bronchial epithelial cells and alveolar macrophages were markedly higher in patients with severe COPD and ex-smokers than in never smokers (p<0.02). Exposure of cultured primary bronchial epithelial cells to smoke resulted in increased expression of both leptin and Ob-Rb (p<0.05). Leptin induced phosphorylation of STAT3 in both NCI-H292 and A549 cells. Conclusions: Leptin expression is increased in bronchial epithelial cells and alveolar macrophages of ex-smokers with or without severe COPD compared with never smokers. A functional leptin signalling pathway is present in lung epithelial cells.

Pro-inflammatory mechanisms of muscarinic receptor stimulation in airway smooth muscle

BackgroundAcetylcholine, the primary parasympathetic neurotransmitter in the airways, plays an important role in bronchoconstriction and mucus production. Recently, it has been shown that acetylcholine, by acting on muscarinic receptors, is also involved in airway inflammation and remodelling. The mechanism(s) by which muscarinic receptors regulate inflammatory responses are, however, still unknown.MethodsThe present study was aimed at characterizing the effect of muscarinic receptor stimulation on cytokine secretion by human airway smooth muscle cells (hASMc) and to dissect the intracellular signalling mechanisms involved. hASMc expressing functional muscarinic M2 and M3 receptors were stimulated with the muscarinic receptor agonist methacholine, alone, and in combination with cigarette smoke extract (CSE), TNF-α, PDGF-AB or IL-1β.ResultsMuscarinic receptor stimulation induced modest IL-8 secretion by itself, yet augmented IL-8 secretion in combination with CSE, TNF-α or PDGF-AB, but not with IL-1β. Pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely normalized the effect of methacholine on CSE-induced IL-8 secretion, whereas PMA, a PKC activator, mimicked the effects of methacholine, inducing IL-8 secretion and augmenting the effects of CSE. Similar inhibition was observed using inhibitors of IκB-kinase-2 (SC514) and MEK1/2 (U0126), both downstream effectors of PKC. Accordingly, western blot analysis revealed that methacholine augmented the degradation of IκBα and the phosphorylation of ERK1/2 in combination with CSE, but not with IL-1β in hASMc.ConclusionsWe conclude that muscarinic receptors facilitate CSE-induced IL-8 secretion by hASMc via PKC dependent activation of IκBα and ERK1/2. This mechanism could be of importance for COPD patients using anticholinergics.

Interleukin 13 Exposure Enhances Vitamin D-Mediated Expression of the Human Cathelicidin Antimicrobial Peptide 18/LL-37 in Bronchial Epithelial Cells

ABSTRACT Vitamin D is an important regulator of the expression of antimicrobial peptides, and vitamin D deficiency is associated with respiratory infections. Regulating expression of antimicrobial peptides, such as the human cathelicidin antimicrobial peptide 18 (hCAP18)/LL-37, by vitamin D in bronchial epithelial cells requires local conversion of 25(OH)-vitamin D3 (25D3) into its bioactive metabolite, 1,25(OH)2-vitamin D3 (1,25D3), by CYP27B1. Low circulating vitamin D levels in childhood asthma are associated with more-severe exacerbations, which are often associated with infections. Atopic asthma is accompanied by Th2-driven inflammation mediated by cytokines such as interleukin 4 (IL-4) and IL-13, and the effect of these cytokines on vitamin D metabolism and hCAP18/LL-37 expression is unknown. Therefore, we investigated this with well-differentiated bronchial epithelial cells. To this end, cells were treated with IL-13 with and without 25D3, and expression of hCAP18/LL-37, CYP27B1, the 1,25D3-inactivating enzyme CYP24A1, and vitamin D receptor was assessed by quantitative PCR. We show that IL-13 enhances the ability of 25D3 to increase expression of hCAP18/LL-37 and CYP24A1. In addition, exposure to IL-13 resulted in increased CYP27B1 expression, whereas vitamin D receptor (VDR) expression was not significantly affected. The enhancing effect of IL-13 on 25D3-mediated expression of hCAP18/LL-37 was further confirmed using SDS-PAGE Western blotting and immunofluorescence staining. In conclusion, we demonstrate that IL-13 induces vitamin D-dependent hCAP18/LL-37 expression, most likely by increasing CYP27B1. These data suggest that Th2 cytokines regulate the vitamin D metabolic pathway in bronchial epithelial cells.

IL-4 and IL-13 exposure during mucociliary differentiation of bronchial epithelial cells increases antimicrobial activity and expression of antimicrobial peptides

The airway epithelium forms a barrier against infection but also produces antimicrobial peptides (AMPs) and other inflammatory mediators to activate the immune system. It has been shown that in allergic disorders, Th2 cytokines may hamper the antimicrobial activity of the epithelium. However, the presence of Th2 cytokines also affects the composition of the epithelial layer which may alter its function. Therefore, we investigated whether exposure of human primary bronchial epithelial cells (PBEC) to Th2 cytokines during mucociliary differentiation affects expression of the human cathelicidin antimicrobial protein (hCAP18)/LL-37 and human beta defensins (hBD), and antimicrobial activity.PBEC were cultured at an air-liquid interface (ALI) for two weeks in the presence of various concentrations of IL-4 or IL-13. Changes in differentiation and in expression of various AMPs and the antimicrobial proteinase inhibitors secretory leukocyte protease inhibitor (SLPI) and elafin were investigated as well as antimicrobial activity.IL-4 and IL-13 increased mRNA expression of hCAP18/LL-37 and hBD-2. Dot blot analysis also showed an increase in hCAP18/LL-37 protein in apical washes of IL-4-treated ALI cultures, whereas Western Blot analysis showed expression of a protein of approximately 4.5 kDa in basal medium of IL-4-treated cultures. Using sandwich ELISA we found that also hBD-2 in apical washes was increased by both IL-4 and IL-13. SLPI and elafin levels were not affected by IL-4 or IL-13 at the mRNA or protein level. Apical wash obtained from IL-4- and IL-13-treated cultures displayed increased antimicrobial activity against Pseudomonas aeruginosa compared to medium-treated cultures. In addition, differentiation in the presence of Th2 cytokines resulted in increased MUC5AC production as has been shown previously.These data suggest that prolonged exposure to Th2 cytokines during mucociliary differentiation contributes to antimicrobial defence by increasing the expression and release of selected antimicrobial peptides and mucus.

The effect of asthma therapeutics on signalling and transcriptional regulation of airway smooth muscle function

SCOPE OF THE REVIEW Our knowledge of the multifunctional nature of airway smooth muscle (ASM) has expanded rapidly in the last decade, but the underlying molecular mechanisms and how current therapies for obstructive airway diseases, such as asthma and chronic obstructive pulmonary disease (COPD), affect these are still being elucidated. Our current knowledge has built on the pharmacology of human ASM contraction and relaxation established prior to that and which is reviewed in detail elsewhere in this issue. The advent of methods to isolate and culture ASM cells, especially human ASM cells, has made it possible to study how they may contribute to airway remodelling through their synthetic, proliferative, and migratory capacities. Now the underlying molecular mechanisms of ASM growth factor secretion, extracellular matrix (ECM) production, proliferation and migration, as well as contraction and relaxation, are being determined. A complex network of signalling pathways leading to gene transcription in ASM cells permits this functional plasticity in healthy and diseased airways. This review is an overview of the effects of current therapies, and some of those in development, on key signalling pathways and transcription factors involved in these ASM functions.

Playing a dirty trick on airway smooth muscle: house dust mite does it again

Increased airway smooth muscle (ASM) mass is a hallmark of asthma and is thought to be the main contributor to the increased airway hyperresponsiveness seen in asthmatics [1]. This increase is related to disease severity (reviewed in [2]) and caused by hyperplasia alone or in combination with hypertrophy, depending on the generation of airways [3, 4]. Why and how ASM mass is increased is incompletely understood, but in vitro studies have shown that ASM cells isolated from asthmatics proliferate faster in culture [5]. In addition, cultured patient cells produce more chemokines and an altered array of extracellular matrix proteins compared with those of healthy individuals [6, 7]. In vitro studies have shown that upon isolation, ASM cells display contractile function, but they rapidly change phenotype ( i.e. acquire proliferative and synthetic capacity) and become noncontractile during culture under serum-rich conditions [8]. When these synthetic–proliferative cells are serum-deprived for a prolonged period of time, a small population (so-called “hypercontractile” cells) starts re-expressing contractile protein genes, such as ACTA2 (encoding α-smooth muscle actin (α-SMA)) [9]. This dedifferentiation/modulation and differentiation/maturation of ASM cells is referred to as phenotype switching or phenotypic plasticity, and although this phenomenon clearly exists in vitro , we do not know whether this plays a role in thickening of the ASM layer. The increase in ASM proliferation in asthma is thought to be associated with decreased levels of CCAAT enhancer protein (c/EBP)α (encoded by the CEBPA gene), a crucial controller of cell cycle progression, differentiation and inflammation. The biological effects of this protein are exerted by the full-length (p42) isoform and counteracted by a shorter isoform (p30), which is formed after translation of the protein. Previously, it was shown that ASM from asthmatics has …

Role of purinergic receptors in the activation of human airway smooth muscle cells by the antimicrobial peptide LL-37

Inflammatory cells that infiltrate and surround the airway smooth muscle (ASM) layer express antimicrobial peptides including the cathelicidin LL-37. LL-37 has been shown to activate epithelial cells by transactivation of the epidermal growth factor receptor (EGFR). Previously, we have shown that LL-37-induced IL-8 release by ASM cells was not dependent on either formyl peptide receptors or the EGFR (ATS 2005). In monocytes LL-37 induces processing of IL-1β through activation of the purinergic P2X7 receptor. Therefore, the aim of our study was to evaluate the role of purinergic receptors in LL-37-induced activation of ASM cells, and to explore the involvement of several intracellular signalling pathways. ASM cells were cultured and serum-deprived 24 hours before stimulation with LL-37 (10 μg·ml−1). The purinergic receptor antagonist suramin and inhibitors of ERK1/2, p38, Src and PI3K were preincubated for one hour. ERK1/2 phosphorylation was assessed by Western Blot, and IL-8 release was determined in supernatants using a sandwich ELISA. RT-PCR was performed for P2X7 on untreated ASM cells. LL-37 induced ERK1/2 phosphorylation and IL-8 release; both were inhibited by suramin (IL-8: 86%). Inhibitors of ERK1/2, p38 and Src signalling also reduced LL-37-induced IL-8 release (by 67%, 63% and 76%, respectively), suggesting a role for these pathways. P2X7 mRNA was expressed in ASM cells. These data show that LL-37-induced IL-8 release is mediated via purinergic receptors, ERK1/2 activation, p38 and Src signalling. Our PCR data are in line with the hypothesis that also in ASM P2X7 is the purinergic receptor involved in LL-37 signalling, although this needs further investigation.