Cholinergic receptor activation on epithelia protects against cytokine-induced barrier dysfunction

Aim: Various types of cholinergic receptors are expressed on intestinal epithelia. Their function is not completely understood. We hypothesize that cholinergic receptor activation on epithelium may serve a protective function in cytokine-induced barrier dysfunction. Methods: The effect of cholinergic receptor activation on cellular barrier function in epithelial cells was assessed by measuring electrical impedance, and by determining para-cellular transport in transwell experiments. Cell lysates treated with cytokine and/or cholinergic agonists were analysed for cyto- and chemokine production, and tight junction (TJ) protein rearrangement was assessed. Primary colonic epithelial cells were isolated from surgically resected colon tissue of patients with inflammatory bowel disease. Results: IL-1 beta induced production of chemokines (CXCL-1, CXCL-10, IL-8, CCL-7) and led to a rearrangement of TJ proteins (occludin and ZO-1). This response was inhibited by pre-treatment with muscarinic, rather than nicotinic, acetylcholine receptor agonists. Treatment with IL-1 beta enhanced paracellular permeability (4kD dextran) and reduced impedance across the monolayer, which was counteracted by pre-incubation with acetylcholine, or muscarinic receptor agonist bethanechol. The protective effect of acetylcholine was antagonized by atropine, underscoring muscarinic receptor involvement. IL-1b induced transcription of myosin light chain kinase and phosphorylation of myosin light chain, and this cytokine-induced phosphorylation of MLC was inhibited by muscarinic receptor agonists. Furthermore, in epithelial cells from resection material of patients with Crohn's disease and ulcerative colitis, high expression of CXCL-8 was associated with a reduced choline acetyl transferase expression, suggesting an aberrant epithelial production of ACh in inflammatory context. Conclusion: Acetylcholine acts on muscarinic receptors on epithelial cells to maintain epithelial barrier function under inflammatory conditions.