Nicotine Attenuates Activation of Tissue Resident Macrophages in the Mouse Stomach through the β2 Nicotinic Acetylcholine Receptor

Background: The cholinergic anti-inflammatory pathway is an endogenous mechanism by which the autonomic nervous system attenuates macrophage activation via nicotinic acetylcholine receptors (nAChR). This concept has however not been demonstrated at a cellular level in intact tissue. To this end, we have studied the effect of nicotine on the activation of resident macrophages in a mouse stomach preparation by means of calcium imaging. Methods: Calcium transients ([Ca2+](i)) in resident macrophages were recorded in a mouse stomach preparation containing myenteric plexus and muscle layers by Fluo-4. Activation of macrophages was achieved by focal puff administration of ATP. The effects of nicotine on activation of macrophages were evaluated and the nAChR involved was pharmacologically characterized. The proximity of cholinergic nerves to macrophages was quantified by confocal microscopy. Expression of beta 2 and alpha 7 nAChR was evaluated by beta 2 immunohistochemistry and fluorophore-tagged alpha-bungarotoxin. Results: In 83% of macrophages cholinergic varicose nerve fibers were detected at distances <900nm. The ATP induced [Ca2+](i) increase was significantly inhibited in 65% or 55% of macrophages by 100 mu M or 10 mu M nicotine, respectively. This inhibitory effect was reversed by the beta 2 nAChR preferring antagonist dihydro-beta-eryhtroidine but not by hexamethonium (non-selective nAChR-antagonist), mecamylamine (alpha 3 beta 4 nAChR-preferring antagonist), alpha-bungarotoxin or methyllycaconitine (both alpha 7 nAChR-preferring antagonist). Macrophages in the stomach express beta 2 but not alpha 7 nAChR at protein level, while those in the intestine express both receptor subunits. Conclusion: This study is the first in situ demonstration of an inhibition of macrophage activation by nicotine suggesting functional signaling between cholinergic neurons and macrophages in the stomach. The data suggest that the beta 2 subunit of the nAChR is critically involved in the nicotine-induced inhibition of these resident macrophages.