The interaction of tiotropium with long lasting β2-agonists on lung cell function

The major question addressed in this thesis was to find the mechanism(s) by which muscarinic receptors interact with β2-adrenergic receptors in human airway fibroblasts. This question is of importance to understand the molecular biological basis of the clinical observation that blocking the muscarinic receptots, while activating the β2-adernergic receptor allows better symptom control in COPD and asthma then increasing the concentration of a single drug. This knowledge will also help to improve and optimize the action of the two drugs when combined.
The question what is the molecular biological basis of the improved beneficial clinical effects observed in COPD patients treated by a combination of muscarinic receptor inhibitors and long acting β2-aganists became of sepcial interest for asthma therapy after Grainge et al (2011) described that airway remodelling when induced by allergens or cholinergic stimuli was prevented when the patients had inhaled a short acting β2-agonist. This study not only indicated a novel unknown interactive mechanism between the muscarinic receptor and the β2-adrenergic receptor, in addition, it provided for the first time clear in vivo experimental evidence in humans, that asthma associated airway wall remodelling is independent of preceding inflammation and thast it occurs within days and does not need months as indicated by animal models.
In my thesis I provide first evidence, that not only airway smmoth muscle cells, but also human primary lung fibroblasts, isolated from lungs of asthma patients, do secrete more pro-inflammatory cytokines than cells isolated from non-asthmatic patients (including COPD). This disease specific pro-inflammatory response, however, was not occuring under all conditions, but was depnednent on the type of stimulus used. Comparing the effect of three different asthma relevant stimuli, PDGF-BB, IL-1β and TNF-α, it was obvious that TNF-α had a stronger indcutive effect on IL-6 secretion in fibroblasts of asthma patients compared to non-asthmatic cells, while it had a stimulating but not disease specific effect on IL-8 secretion. PDGF-BB had a similar inductive effect on IL-6 secretionin both asthmatic and non-asthmatic firboblasts, while it had a signifcant stronger inducing effect on IL-8 secretion by asthmatic fibroblasts compared to control cells. In contrast, stimulation with IL-1β significantly stronger up-regulated the secretion of IL-6 and IL-8 by control fibroblasts compared to cells of asthma patients.
Carbachole, a muscarinic receptor activator, had no stimulative effect on either cytokine, neither in asthmatic nor in control fibroblasts. However, when combined with especially IL-1β it further increased the cytokine secretion.Therefore, it can be concluded that sub-epithelial fibroblasts in the airway wall represent an additional source of pro-inflammatory cytokines. These initial findings were the reason why the combination of IL-1β with carbachole was used in all sub-seqeunt experiments to investigate the effects of the long acting β2 agonist olodaterol and the muscarinic receptor inhibitor tiotropium on cytokine secretion by fibroblasts.
Both classes of drugs, olodaterol and tiotropium, alone significantly reduced the IL-1β induced secretion of IL-6 and IL-8. When combined their inhibitory effects were only additive. Thus, the therapeutic combination of both classes of drugs may be beneficial, but has to be proven for other compounds.
Our group has provided earlier data that showed the expression of the β2-adrenergic receptor on the cell surface of human lung fibroblasts, but there was no data for the type of muscarinic receptor (MR1-5) was expressed by the cells. Using RT-PCR we showed that the majority of muscarinic receptors expressed by human lung fibroblasts were if type-3 and only little of type-1.
We further investigated the sigballing pathway underlying the anti-inflammatory effect of the β2-agonist and the muscarinic receptor inhibitor. The data showed thatin part the inhibitory mwechanism invloves the increase of intracellular cAMP levels, which is known to mediate the muscle relaxing effect of this class of drugs. However, it was surprising that carbachol overruled the anti-inflammatory effect of the β2-agonist which was not in line with the in vivo results presented by Grainge et al (2011), but would fit with other studies showing only a limited ant-inflammatory effect of β2-agonsists.
However, when the cells were pre-incubated with the muscarinic receptor inhibitor tiotropium for 30 minutes prior to the addition of carbachol the β2-agonist dependent increase of the intracellular cAMP levekl was rescued, followed by activation of the cyclic AMP response element (CREB). These findings may explain why the anti-inflammatory effect of the combined drugs was additive rather than synergistic.
Initially it was assumed that the clinically observed beneficial effect of the combined drugs may result from the interaction of specific G-proteins to which both receptor types are linked (see figure below). However, the obtained data did not indicate any role of G-proteins in the anti-inflammatory effect of neither the β2 adreneregic receptor nor of the muscarinic receptor.
Proposed separation of the anti-inflammatory and anti-proliferative signalling interaction of muscarinic and β2-adrenergic receptors in human lung fibroblasts.
Beside inflammation, airway wall remodelling is characterized by an extensive increase of sub-epithelial fibroblast numbers and extracellular matrix deposition. According to the literature there is no drug in asthma therapy which has a significant reducing effect on airway wall remodelling. In earlier studies our research group had shown that the lack of anti-proliferative effect of at least glucocorticoids is due to the lack of a differentiation/cell cycle control factor, CCAAT enhancer binding protein-α (C/EBP-α), which disease sepcifically missing in airway smooth muscle cells of asthma patients. Unpublished data suggested that at least the β2-agonists, formoterol and salmeterol, have an anti-proliferative effect of 30% reduction in airway smooth muscle cells. The literature reported simliar small effects of anti-proliferative action of β2-agonists and even reported pro-proliferative actions. In contrast to glucocortisoids which depend on C/EBP-α and sub-seqeunt activity of p21(Waf), the anti-proliferative action of β2-agonists invloved p27(Kip). In this thesis, we tested the inhibitory potebtial of olodaterol on fibroblast proliferation induced by PDGF-BB.
At the time of this thesis, there was no data published showing an inhibitory effect of muscarinic receptor inhibitors in regard of fibroblast proliferation.
Olodaterol confirmed an inhibitory effect of β2-agonists on lung fibroblast proliferation, when the cells were stimulated with PDGF-BB. The anti-proliferative effect of olodaterol was dose-dependent and was paralleled by the increase of intracellular cAMP. Based on our earlier data with formoterol and salmeterol we concluded that this beneficial effect applies to all β2-agonists.
Combining olodaterol with the muscarinic receptor inhibitor, however, did not improve tha anti-proliferative effect of the β2-agonist. In contrast the muscarinic receptor inhibitor counteracted the anti-proliferative effect of olodaterol to a certain extend. Surprizingly, neither the anti-proliferative effect of olodaterol nor the counteractive effect of tiotropium could be linked to the rescue of β2-agonist cAMP level increase or to G-protein activity. Importantly, similar results have been recently published by others and suggest a novel anti-proliferative acting signalling pathway for β2-adrenergic receptors through so called β-arrestins, which are involved in muscarinic receptor activity, however, with controversial results. Therefore, no conclusion on the role of β-arrestins as an anti-proliferative proetin in the signalling of combined β2 agonists and muscarinic receptor inhibitors can be made. It would be interesting to screen our samples for the expression of β-arrestins.
The impact of this thesis on the undertsanding of the interaction of β2-adrenergic receptors and muscarinic receptor signalling is as follows:
(i) The intracellular signalling cascades that get activated by the muscarinic receptor-3 or by the β2-adrenergic receptor are not involving G-proteins;
(ii) That combined β2-agonsists and muscarinic receptor inhibitors indeed have beneficial additive anti-inflammatory action which may be due to a rescuing effect of muscrainic receptor inhibition on β2-adrenergic receptor dependent intracellular cAMP activation.
(iii) The combination of the two classes of drugs is not beneficial for airwaywall remodelling based on increased fibrotic lesions, and thus is in line with clinical data. However, proliferation is only one part of remodelling which also includes increased deposition of extracellular matrix.
(iv) Preliminary experiments on the deposition of collagens and fibronectin show that β2-agonsits reduce TGF-β and endothelin induced depsoition of extracelllular matrix components but do not have a general inhibitory effect. Some of the inhibitory effects of β2-agonists on cllagens can be explained by the increase of cAMP, while the inhibitory effect on fibronectin is independent of cAMP. The available data indicates that long actinng β2-agonists (fornmoterol, salmeterol) reduce the deposition of specifically collagen type-I and of fibronectin through a cAMP dependent and a cAMP independent pathway.
In summary, the data obtained in this thesis answered some aspects of the interaction of the two receptor types, but also raised further questions. In addition, it became clear that some of the beneficial anti-inflammatory actions of combined β2-adrenergic receptor agonsists with inhibitors of muscarinic receptors invloves the action of cAMP while others do not. The beneficail anti-inflammatory action of combined β2-adrenergic receptor agonsists with inhibitors of muscarinic receptors is clear, but the net-effect of the two drugs on tissue remodelling has to be further investigated.
Finally, it became clear, that we need to better understand how β2-adrenergic receptors and muscarinic receptors mediate their signals to the cells, it seems that we do not yet know all the details and novel mechanisms will be described soon.