Abstract
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.
Highlights
Acetylcholine, the primary parasympathetic neurotransmitter in the airways, plays an important role in bronchoconstriction and mucus production
Muscarinic receptor stimulation facilitates cytokine secretion induced by cigarette smoke extract (CSE), TNF-a and platelet-derived growth factor-AB (PDGF-AB) Recently, it has been reported that stimulation of muscarinic receptors induces the release of IL-8 from human bronchial epithelial cells and facilitates the release of IL-8 from human airway smooth muscle cells (hASMc) induced by CSE [8,19]
We evaluated the pro-inflammatory properties of muscarinic receptor stimulation in hASMc, alone and in concerted action with CSE (5%), PDGF-AB (30 ng/mL), TNF-a (1 ng/mL) or IL-1b (1 ng/mL) (Figure 1)
Summary
Acetylcholine, the primary parasympathetic neurotransmitter in the airways, plays an important role in bronchoconstriction and mucus production. Acetylcholine, the primary parasympathetic neurotransmitter in the airways plays an important role in COPD, by regulating bronchoconstriction and mucus production [5]. It has been reported that airway vascular leakage is mediated by muscarinic receptors [13] These findings suggest a role in pro-inflammatory responses for muscarinic receptors. It is still undefined what the potential anti-inflammatory effects of muscarinic antagonists are in the lungs of patients with COPD [14], which is in part due to the unknown mechanisms behind the regulation of inflammatory responses by muscarinic receptors
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