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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.

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.

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.

Recombination of germ-line TCR alpha and beta genes generates polypeptide receptors for MHC-peptide. Antigen 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 of T-cell receptor alpha and beta polypeptides from T-cell clones recognizing VP22 amino acids 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. 19% to 72% of tetramer-binding cells in PBMC stained ex vivo with a TCRBV12 mAb. Three alpha chain and three beta chain public T-cell receptor 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 interferon-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 alphaherpesvirus epitope converges on preferred T-cell receptor sequences with relatively constant functional avidity.