Muscarinic receptors in airway smooth muscle:roles in inflammation and remodelling

Anticholinergica, die muscarinereceptoren blokkeren in de luchtwegen, vormen al jaren de hoeksteen van de behandeling van luchtwegvernauwing bij COPD. Tjitske Oenema ontdekte echter dat deze anticholinergica ook nog een belangrijke gunstige rol blijken te spelen bij ontstekingsprocessen en structurele veranderingen in de luchtweg-gladde spier. Oenema ontdekte namelijk enkele nog niet eerder bekende schadelijke effecten die gepaard gaan met de activatie van muscarinereceptoren. Zo blijken geactiveerde muscarinereceptoren de IL-8 productie als gevolg van sigarettenrook - een van de belangrijkste veroorzakers van COPD - te versterken. Ook toonde Oenema aan dat muscarinereceptoren de effecten van de groeifactor TGF-β, die verhoogd tot expressie wordt gebracht bij COPD, op contractiele eiwitexpressie en celproliferatie van luchtweg-gladde spiercellen kan versterken. Beide processen dragen potentieel bij aan de chronische progressie van COPD. Het effect van muscarinereceptoractivatie op de expressie van contractiele eiwitten geïnduceerd door TGF-β is het gevolg van activatie van specifieke intracellulaire signaaltransductieroutes waarin het enzym GSK-3 een centrale rol speelt. Langdurige blootstelling aan TGF-β induceert luchtweg-gladde spiercelproliferatie door verhoogde depositie van de matrixeiwitten (bindweefseleiwitten) collageen I en fibronectine en hun interactie met integrinereceptoren op de spiercel. Muscarine M2 receptoren versterken deze respons door de productie van fibronectine te versterken. Dit is de eerste aanwijzing dat muscarine-M2-receptoren betrokken zijn bij structurele veranderingen in gladde spiercellen in de luchtweg. Ten slotte ontwikkelde Oenema een in vitro model, gebruik makend van dun gesneden cavialongcoupes om structurele veranderingen van de luchtwegen te bestuderen. Met dit model toonde zij aan dat de constrictie van de luchtwegen door muscarinereceptoractivatie de vrijzetting van TGF-β induceert, wat structurele luchtwegveranderingen bevordert. Samengevat tonen de resultaten van Oenema’s proefschrift een nieuwe rol aan voor muscarinereceptoren bij ontsteking en structurele veranderingen van de luchtwegen. Deze mechanismen zijn mogelijk van groot belang bij de behandeling van COPD patiënten met anticholinergica.

A new perspective on muscarinic receptor antagonism in obstructive airways diseases

<p>Acetylcholine has traditionally only been regarded as a neurotransmitter of the parasympathetic nervous system, causing bronchoconstriction and mucus secretion in asthma and COPD by muscarinic receptor activation on airway smooth muscle and mucus-producing cells. Recent studies in experimental models indicate that muscarinic receptor stimulation in the airways also induces pro-inflammatory, pro-proliferative and pro-fibrotic effects, which may involve activation of airway structural and inflammatory cells by neuronal as well as non-neuronal acetylcholine. In addition, mechanical changes caused by muscarinic agonist-induced bronchoconstriction may be involved in airway remodeling. Crosstalk between muscarinic receptors and beta(2)-adrenoceptors on airway smooth muscle causes a reduced bronchodilator response to beta(2)-agonists, and a similar mechanism could possibly apply to the poor inhibition of inflammatory and remodeling processes by these drugs. Collectively, these findings provide novel perspectives for muscarinic receptor antagonists in asthma and COPD, since these drugs may not only acutely affect cholinergic airways obstruction, but also have important beneficial effects on beta(2)-agonist responsiveness, airway inflammation and remodeling. The clinical relevance of these findings is presently under investigation and starting to emerge.</p>

Precision cut lung slices:An in vitro model to study the role of Tgfβ1 and muscarinic receptors in airway remodeling

RATIONALE: Airway remodelling in chronic asthma is characterized by thickening of the airway wall, including thickening of the smooth muscle layer. Although studies using cellular and molecular models have provided important insights into the mechanisms that underpin airway remodelling, limitations of these models include the prolonged cell culture and the loss of the multicellular context of the airway wall. The purpose of this study was to evaluate precision cut lung slices as an in vitro model to study airway remodeling. Lung slices have all cell types in their natural matrix and largely intact tissue architecture. Previously, we showed that TGFß1 and the muscarinic receptor agonist methacholine (MCh) increased the expression of contractile phenotype markers in cultured human airway smooth muscle cells. Here, we studied the effects of these stimuli on airway remodeling in lung slices. METHODS: Lung slices, obtained from guinea pigs after filling the lungs with 1.5% agarose, were prepared and cultured in the absence or presence of increasing concentrations of TGFß1 (0.1-2 ng/mL) or MCh (10 μM). Slices were tested for viability using a mitochondrial reduction assay. Protein expression was quantified by Western blot and visualized by confocal fluorescence microscopy. RESULTS: Initial experiments indicated that lung slices could be cultured for at least 48 hours without loss of cellular viability. Therefore, in subsequent experiments protein expression and localization was determined in lung slices cultured up to 48h. Western blot analyses showed a concentration-dependent and time-dependent increase in the expression of the contractile proteins myosin, calponin and α-smooth muscle (sm)-actin in response to TGFß1. Additionally, myosin, calponin and α-sm-actin protein expression were increased in response to MCh. Confocal fluorescence microscopy visualized the 3D architecture of airways and parenchymal tissue up to ~100 μm depth and showed that the treatment with TGFß1 and MCh augmented α-sm-actin expression predominantly in the small airways and peribronchiolar tissue. CONCLUSIONS: Collectively, precision cut lung slices are a suitable model to study mechanisms of airway remodeling in vitro. Limitations of the model include the relatively short window in which slices can be kept in culture compared to cell culture, whereas its strengths include the multicellular architecture and the possibility to study protein localization in a 3D context. This suggests that future studies using precision cut lung slices could be used to extrapolate results from studies in isolated cells to more physiologically intact systems

Public TCR use by herpes simplex virus-2-specific human CD8 CTLs.

Recombination of germline TCR alpha and beta genes generates polypeptide receptors for MHC peptide. Ag exposure during long-term herpes simplex infections may shape the T cell repertoire over time. We investigated the CD8 T cell response to HSV-2 in chronically infected individuals by sequencing the hypervariable regions encoding TCR alpha and beta polypeptides from T cell clones recognizing virion protein 22 aa 49-57, an immunodominant epitope. The most commonly detected TCRBV gene segment, found in four of five subjects and in 12 of 50 independently derived T cell clones, was TCRBV12-4. Nineteen to seventy-two percent of tetramer-binding cells in PBMCs were stained ex vivo with a TCRBV12 mAb. Three alpha-chain and three beta-chain public TCR sequences were shared between individuals. Public heterodimers were also detected. Promiscuous pairing of a specific TCRVA1-1 sequence with several different TCRB polypeptides was observed, implying a dominant structural role for the TCRA chain for these clonotypes. Functional avidity for cytotoxicity and IFN-gamma release was relatively invariant, except for one subject with both high avidity and unique TCR sequences and lower HSV-2 shedding. These data indicate that the CD8 response to a dominant alpha-herpesvirus epitope converges on preferred TCR sequences with relatively constant functional avidity

Precision cut lung slices: A novel in vitro model to demonstrate that bronchoconstriction is a key player in airway remodelling

Bronchoconstriction has been proposed to underlie airway remodelling. However, the mechanisms involved are still poorly understood. An important contributor to airway remodelling, in particular airway smooth muscle remodelling is the multifunctional cytokine TGF-β, which can facilitate airway smooth muscle maturation by inducing the expression of contractile proteins. The aim of this study was to develop an in vitro model which allows investigation of mechanisms involved in the bronchoconstriction-induced airway remodelling. To this aim, precision cut lung slices of guinea pigs were used. Lung slices were viable for at least 2 days. Stimulation with either methacholine or TGF-β1 augmented the expression of contractile proteins makers including sm-myosin, sm-β-actin and calponin after 2 days. The expression of sm-myosin was particularly enhanced in the peripheral airways ( 400 μM). Inhibition of actin polymerization by Latrunculin A or of TGF-β receptor kinase by SB431542 prevented the methacholine effects and the effects of other bronchoconstricting agents including histamine and KCl on contractile protein expression. This indicates the release of biologically active TGF-β in response to bronchoconstriction, ensuing contractile protein expression and thus airway smooth muscle remodelling.The anticholinergic tiotropium bromide inhibited the methacholineinduced contractile protein expression in a concentration-dependent manner. Collectively, bronchoconstriction induces the release of biologically active TGF-β and promotes remodelling. Muscarinic receptor induced remodelling can be counteracted by the use of the anticholinergic tiotropium bromide. Precision cut lung slices are a valid in vitro model to study mechanisms involved in airway remodelling induced for example, by bronchoconstriction

Muscarinic receptor stimulation augments TGF-beta(1)-induced contractile protein expression by airway smooth muscle cells

Oenema TA, Smit M, Smedinga L, Racke K, Halayko AJ, Meurs H, Gosens R. Muscarinic receptor stimulation augments TGF-beta(1)-induced contractile protein expression by airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 303: L589-L597, 2012. First published August 3, 2012; doi:10.1152/ajplung.00400.2011.-Acetylcholine (ACh) is the primary parasympathetic neurotransmitter in the airways. Recently, it was established that ACh, via muscarinic receptors, regulates airway remodeling in animal models of asthma and chronic obstructive pulmonary disease (COPD). The mechanisms involved are not well understood. Here, we investigated the functional interaction between muscarinic receptor stimulation and transforming growth factor (TGF)-beta(1) on the expression of contractile proteins in human airway smooth muscle (ASM) cells. ASM cells expressing functional muscarinic M-2 and M-3 receptors were stimulated with methacholine (MCh), TGF-beta(1), or their combination for up to 7 days. Western blot analysis revealed a strong induction of sm-alpha-actin and calponin by TGF-beta(1), which was increased by MCh in ASM cells. Immunocytochemistry confirmed these results and revealed that the presence of MCh augmented the formation of sm-alpha-actin stress fibers by TGF-beta(1). MCh did not augment TGF-beta(1)-induced gene transcription of contractile phenotype markers. Rather, translational processes were involved in the augmentation of TGF-beta(1)-induced contractile protein expression by muscarinic receptor stimulation, including phosphorylation of glycogen synthase kinase-3 beta and 4E-binding protein 1, which was enhanced by MCh. In conclusion, muscarinic receptor stimulation augments functional effects of TGF-beta(1) in human ASM cells on cellular processes that underpin ASM remodeling in asthma and COPD

Delivery system for budesonide based on lipid-DNA.

Budesonide is a hydrophobic glucocorticoid with high anti-inflammatory activity for the treatment of asthma, inflammatory bowel disease and rheumatoid arthritis. A micellar drug-delivery system based on lipid-DNA may provide a strategy to maximize its drug efficacy and reduce adverse effects. In this work, we report the use of lipid-DNAA (UU11mer), featuring two hydrophobic alkyl chains and forming micelles at a comparatively low critical micelle concentration, to render budesonide water-soluble with a high loading capacity (LC). The inhibition of interleukin-8 (IL-8) release shows that the new delivery system retains the inhibitory activity in cell-based assays. In conclusion, this research provides a novel approach to formulate and administer budesonide in a non-invasive manner, which dramatically improves its water-solubility while retaining its bioavailability

De novo synthesis of beta-catenin via H-Ras and MEK regulates airway smooth muscle growth

beta-Catenin is a component of adherens junctions that also acts as a transcriptional coactivator when expressed in the nucleus. Growth factors are believed to regulate the nuclear expression of beta-catenin via inactivation of glycogen synthase kinase 3 (GSK-3) by phosphorylation, resulting in increased beta-catenin protein stability. Here, we report on a novel pathway that regulates the expression and nuclear presence of beta-catenin. In proliferating human airway smooth muscle cells, we observed increased expression of beta-catenin, which was required for proliferation. Interestingly, increased beta-catenin expression was accompanied by an increase in beta-catenin mRNA and was independent of beta-catenin liberation from the plasma membrane, suggesting a role for de novo synthesis. This was confirmed using actinomycin D and cycloheximide, which abrogated the induction and nuclear localization of beta-catenin protein. GSK-3 inhibition using SB216763 failed to regulate beta-catenin mRNA. However, expression of dominant negative H-Ras or pharmacological inhibition of MEK reduced serum and TGF-beta-induced beta-catenin mRNA and protein. Collectively, these data indicate that beta-catenin is an important signaling intermediate in airway smooth muscle growth and that its cellular accumulation and nuclear localization require de novo protein synthesis effected, in part, via H-Ras and MEK.-Gosens, R., Baarsma, H. A., Heijink, I. H., Oenema, T. A., Halayko, A. J., Meurs, H., Schmidt, M. De novo synthesis of beta-catenin via H-Ras and MEK regulates airway smooth muscle growth. FASEB J. 24, 757-768 (2010). www.fasebj.or