In the present study, we demonstrate that muscarinic receptors stimulate the secretion of the pro-inflammatory cytokine IL-8 from hASMc, and augment the response induced by TNF-α, CSE and PDGF-AB. Furthermore, we dissected the underlying mechanism of the synergistic IL-8 production. To permit the release of the pro-inflammatory cytokine IL-8 after activation of the muscarinic receptors and CSE, activation of PKC is required, which is followed by the breakdown of IκBα. In parallel, the activation of PKC leads to the stimulation of MEK1/2 inducing the phosphorylation of ERK1/2. Both pathways regulate IL-8 secretion, which, as previously described, is dependent on NF-κB and AP-1 IL-8 promoter activation .
Our current and previously published data  indicate that the activation of muscarinic receptors in hASMc facilitates the secretion of the pro-inflammatory cytokines IL-6 and IL-8 in combination with CSE and pro-inflammatory cytokines. Muscarinic receptor stimulation also promoted IL-8 secretion by itself, though only to a relatively minor extent. This suggests that the effects of muscarinic receptor stimulation are relevant primarily in a pro-inflammatory microenvironment. In support, functional muscarinic receptors are expressed on the majority of inflammatory cells . Also, the endogenous muscarinic receptor ligand acetylcholine and its synthesizing enzyme choline acetyltransferase (ChAT) are present in several extraneuronal cell types, including airway epithelial cells, lymphocytes, eosinophils, neutrophils, macrophages, and mast cells [5, 26]. Furthermore, animal models showed that atropine reduces lung inflammation induced by diesel-soot in rats , and that tiotropium bromide inhibits several aspects of airway inflammation and remodeling in ovalbumin-sensitized guinea pigs, but has little effect on inflammatory cell counts in saline challenged controls [11, 27]. Additionally, it has been reported that carbachol, by activation of muscarinic receptors, is able to increase inflammatory gene expression in ASM, including IL-6, IL-8 and cyclooxygenase-2 (COX-2) . Furthermore, acetylcholine (ACh) can induce leukotriene B4 (LTB4) release from sputum COPD cells , also indicating a regulatory role for ACh in inflammatory cells. Taken together, this indicates that acetylcholine is importantly involved in the regulation of pro-inflammatory responses. Our current results provide new insights as we demonstrate that the activation of muscarinic receptors interacts with several cytokines and growth factors, in particular with TNF-α, PDGF-AB and CSE to enhance their inflammatory response in hASMc.
HASMc produce a variety inflammatory mediators [15, 16, 29]. This suggests an important role for ASM in inflammatory responses in COPD. Indeed, hASMc are a source of chemokines and cytokines that play a role in chronic pulmonary diseases like COPD and asthma, including IL-8 and IL-6. The levels of IL-8 are correlated with the degree of neutrophilic inflammation and are increased in sputum in COPD patients [3, 30]. Several pro-inflammatory stimuli, including IL-17 [31–33], gram-positive and gram-negative bacteria , β-tryptase , IL-1β  and TNF-α  are able to induce IL-8 secretion from human ASM. Moreover, CSE synergizes with TNF-α to enhance IL-8 secretion by ASM . We previously demonstrated that CSE and muscarinic M3 receptor stimulation leads to a synergistic increase in IL-8 secretion by hASMc , which as demonstrated in this study, is dependent on downstream signalling to PKC and the IκBα/NF-κB and MEK/ERK1/2 pathways. Nicotinic receptors and muscarinic M2 receptors are not involved in this synergism, as gallamine had no effect on IL-8 release induced by either CSE or MCh . This indicates that acetylcholine may also play an important role in the inflammatory/immunomodulatory processes driven by human ASM.
Using the PKC inhibitor GF109203X, we demonstrate that the synergism of MCh and CSE-induced IL-8 secretion is mediated by PKC in hASMc. In fact, activation of PKC was sufficient to induce synergistic IL-8 secretion in combination with CSE, which was confirmed by the use of the PKC activator, PMA. These observations correspond with an earlier study from our group demonstrating that MCh augments PDGF-induced cell proliferation via the activation of PKC  and appear to suggest that muscarinic M3 receptors exert their facilitatory effects on remodeling and inflammation to an important extent via the activation of PKC. Downstream, we demonstrated that PKC is able to induce the activation of IκBα/NF-κB and MEK/ERK1/2 pathways in hASMc and that these pathways are involved in the secretion of IL-8 induced by the co-stimulation of muscarinic receptors and CSE. Interestingly, the co-stimulation with CSE and MCh appeared required to reveal the importance of PKC, as stimulation with either CSE or MCh alone was not sufficient to demonstrate an involvement of PKC. This indicates that PKC stimulation by MCh is not sufficient to induce an IL-8 or IL-6 response by itself, but augments pro-inflammatory signalling to NF-κB and ERK1/2 induced by CSE. However, synergistic functional interactions with IL-1β, an important cytokine in COPD pathogenesis , were not observed, both for IL-8 secretion and for activation of the signalling pathways investigated, indicating that the mechanism of the synergistic interaction is stimulus specific. Lower concentrations of IL-1β were also tested and were found to be similarly unaffected by MCh (data not shown).
The combination of MCh and CSE likely triggers PKC to activate IKK-2. This kinase allows the phosphorylation and degradation of IκBα leading to the translocation of NF-κB into the nucleus to regulate NF-κB gene transcription . Furthermore, PKC has been shown to be critically involved in the activation of the ERK1/2 pathway in human aortic smooth muscle cells . PKC induces the phosphorylation of Raf-1, an upstream regulator of ERK1/2 activation, which is followed by the regulation of AP-1 dependent gene transcription. The IL-8 gene contains both NF-κB and AP-1 binding sites in its promoter region . Epithelial cells are also able, to induce IL-8 secretion through the activation of ERK1/2 and NF-κB in response to pro-inflammatory stimuli, including acetylcholine [8, 39, 40]. Taken together, these findings and our previous findings  indicate that the synergism between muscarinic M3 receptors and CSE is mediated by PKC dependent activation of the downstream pathways NF-κB and ERK1/2, to induce the secretion of IL-8.
It is unclear whether the pro-inflammatory effects of muscarinic receptor stimulation and CSE, as observed in our current work, are relevant to the COPD patient. Nonetheless, several clinical studies demonstrated that short-term therapy with tiotropium bromide improves airflow and hyperinflation [41, 42]. Moreover, long-term use (up to 6 to 12 months) of this anticholinergic drug improved exercise tolerance, quality of life, rates of dyspnoea but also the exacerbation frequency in COPD patients, which are associated with periods of increased inflammatory cell influx [41, 43]. The Understanding Potential Long-Term Impacts on Function with Tiotropium (UPLIFT) study concluded that COPD patients treated with tiotropium bromide during a 4-year period improved their quality of life, frequency of exacerbations and lung function, but tiotropium bromide did not reduce the decline in FEV1 over the treatment period . Nonetheless, in a subgroup of COPD patients of the UPLIFT study, which were not on other controller medication, a reduction in the accelerated FEV1 decline was observed in the tiotropium bromide arm (post-hoc analysis of the UPLIFT study ). This was also observed in the subgroup of stage II COPD patients . Collectively, besides the well described bronchodilatory effects, these findings suggest additional, non-bronchodilator properties for tiotropium bromide . An anti-inflammatory role for anticholinergics is in agreement with animal and cell culture studies showing a role for acetylcholine in cell proliferation, extracellular matrix protein secretion and inflammation [5, 46, 47] and with our present findings showing that the inflammatory response induced by CSE, TNF-α and PDGF-AB can be augmented by muscarinic receptor stimulation in hASMc. It should be emphasized, however, that the hypothesis that tiotropium bromide may exert anti-inflammatory effects in COPD patients still needs to be tested in clinical studies.