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Nicotine attenuates activation of tissue resident macrophages in the mouse stomach through the β2 nicotinic acetylcholine receptor.

Nemethova A, Michel K, Gomez-Pinilla PJ, Boeckxstaens GE, Schemann M - PLoS ONE (2013)

Bottom Line: 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.This inhibitory effect was reversed by the β2 nAChR preferring antagonist dihydro-β-eryhtroidine but not by hexamethonium (non-selective nAChR-antagonist), mecamylamine (α3β4 nAChR-preferring antagonist), α-bungarotoxin or methyllycaconitine (both α7 nAChR-preferring antagonist).

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Experimental Medicine, Translational Research Center for Gastrointestinal Disorders (TARGID), University of Leuven, Leuven, Belgium.

ABSTRACT

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 ([Ca(2+)]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 β2 and α7 nAChR was evaluated by β2 immunohistochemistry and fluorophore-tagged α-bungarotoxin.

Results: In 83% of macrophages cholinergic varicose nerve fibers were detected at distances <900 nm. The ATP induced [Ca(2+)]i increase was significantly inhibited in 65% or 55% of macrophages by 100 µM or 10 µM nicotine, respectively. This inhibitory effect was reversed by the β2 nAChR preferring antagonist dihydro-β-eryhtroidine but not by hexamethonium (non-selective nAChR-antagonist), mecamylamine (α3β4 nAChR-preferring antagonist), α-bungarotoxin or methyllycaconitine (both α7 nAChR-preferring antagonist). Macrophages in the stomach express β2 but not α7 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 β2 subunit of the nAChR is critically involved in the nicotine-induced inhibition of these resident macrophages.

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Pharmacology of the inhibitory effect of nicotine on ATP-evoked macrophage activation.A, In presence of hexamethonium (Hexa), mecamylamine (Meca), different concentrations of α-bungarotoxin (ABGT) or different concentrations of methyllycaconitine (MLA), 100 µM nicotine still had a significant inhibitory effect on ATP-evoked [Ca2+]i signals in macrophages (Wilcoxon Signed Rank test, * P < 0.001). B, In contrast, DHBE reversed the inhibitory effect of nicotine (Wilcoxon Signed Rank test, P = 0.156). C, ABGT did not reverse the inhibitory effect of 10 µM nicotine on ATP-evoked macrophage activation (Wilcoxon Signed Rank test, * P < 0.001). Please note that the scatter plots illustrating the inhibitory effect of 10 µM nicotine on ATP responses are identical to those in Figure 2 D. We show them here again as we used the same preparations, but different regions, to test the effect of 100 nM ABGT.For all panels: ATPctr labels the maximum [Ca2+]i amplitude to a control ATP administration. ATPnic labels the maximum [Ca2+]i amplitude to ATP application immediately after a 10 sec nicotine administration 10 minutes after the control ATP application. Numbers in parenthesis indicate number of macrophages / number of preparations (equal to number of animals).
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pone-0079264-g003: Pharmacology of the inhibitory effect of nicotine on ATP-evoked macrophage activation.A, In presence of hexamethonium (Hexa), mecamylamine (Meca), different concentrations of α-bungarotoxin (ABGT) or different concentrations of methyllycaconitine (MLA), 100 µM nicotine still had a significant inhibitory effect on ATP-evoked [Ca2+]i signals in macrophages (Wilcoxon Signed Rank test, * P < 0.001). B, In contrast, DHBE reversed the inhibitory effect of nicotine (Wilcoxon Signed Rank test, P = 0.156). C, ABGT did not reverse the inhibitory effect of 10 µM nicotine on ATP-evoked macrophage activation (Wilcoxon Signed Rank test, * P < 0.001). Please note that the scatter plots illustrating the inhibitory effect of 10 µM nicotine on ATP responses are identical to those in Figure 2 D. We show them here again as we used the same preparations, but different regions, to test the effect of 100 nM ABGT.For all panels: ATPctr labels the maximum [Ca2+]i amplitude to a control ATP administration. ATPnic labels the maximum [Ca2+]i amplitude to ATP application immediately after a 10 sec nicotine administration 10 minutes after the control ATP application. Numbers in parenthesis indicate number of macrophages / number of preparations (equal to number of animals).

Mentions: The activation of macrophages by ATP and the inhibition of the ATP response by 100 µM nicotine was reliably recorded in each of the 21 preparations illustrated in Figure 2F. This allowed us to perform antagonists studies without the need to restudy the inhibitory response in those macrophages treated with the antagonists. Moreover, we thereby reduced the number of recording periods to a level that did not compromise signal strength and guaranteed reproducible ATP responses. In order to study the nAChR-subunits involved in the inhibitory effect of nicotine, we tested five different blockers with known subunit preferences [17] (Figure 3). The inhibitory effect of nicotine on ATP-evoked [Ca2+]i responses was unchanged in the presence of the non-selective ganglionic nAChR antagonist hexamethonium, the α3β4 nAChR-preferring antagonist mecamylamine or α7 nAChR-preferring antagonists α-bungarotoxin and methyllycaconitine (Figure 3A). However, the β2 nAChR-preferring antagonist di-hydro-β-eryhtroidine reversed the inhibitory effect of nicotine on ATP-evoked [Ca2+]i responses in macrophages (Figure 3B).


Nicotine attenuates activation of tissue resident macrophages in the mouse stomach through the β2 nicotinic acetylcholine receptor.

Nemethova A, Michel K, Gomez-Pinilla PJ, Boeckxstaens GE, Schemann M - PLoS ONE (2013)

Pharmacology of the inhibitory effect of nicotine on ATP-evoked macrophage activation.A, In presence of hexamethonium (Hexa), mecamylamine (Meca), different concentrations of α-bungarotoxin (ABGT) or different concentrations of methyllycaconitine (MLA), 100 µM nicotine still had a significant inhibitory effect on ATP-evoked [Ca2+]i signals in macrophages (Wilcoxon Signed Rank test, * P < 0.001). B, In contrast, DHBE reversed the inhibitory effect of nicotine (Wilcoxon Signed Rank test, P = 0.156). C, ABGT did not reverse the inhibitory effect of 10 µM nicotine on ATP-evoked macrophage activation (Wilcoxon Signed Rank test, * P < 0.001). Please note that the scatter plots illustrating the inhibitory effect of 10 µM nicotine on ATP responses are identical to those in Figure 2 D. We show them here again as we used the same preparations, but different regions, to test the effect of 100 nM ABGT.For all panels: ATPctr labels the maximum [Ca2+]i amplitude to a control ATP administration. ATPnic labels the maximum [Ca2+]i amplitude to ATP application immediately after a 10 sec nicotine administration 10 minutes after the control ATP application. Numbers in parenthesis indicate number of macrophages / number of preparations (equal to number of animals).
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Related In: Results  -  Collection

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pone-0079264-g003: Pharmacology of the inhibitory effect of nicotine on ATP-evoked macrophage activation.A, In presence of hexamethonium (Hexa), mecamylamine (Meca), different concentrations of α-bungarotoxin (ABGT) or different concentrations of methyllycaconitine (MLA), 100 µM nicotine still had a significant inhibitory effect on ATP-evoked [Ca2+]i signals in macrophages (Wilcoxon Signed Rank test, * P < 0.001). B, In contrast, DHBE reversed the inhibitory effect of nicotine (Wilcoxon Signed Rank test, P = 0.156). C, ABGT did not reverse the inhibitory effect of 10 µM nicotine on ATP-evoked macrophage activation (Wilcoxon Signed Rank test, * P < 0.001). Please note that the scatter plots illustrating the inhibitory effect of 10 µM nicotine on ATP responses are identical to those in Figure 2 D. We show them here again as we used the same preparations, but different regions, to test the effect of 100 nM ABGT.For all panels: ATPctr labels the maximum [Ca2+]i amplitude to a control ATP administration. ATPnic labels the maximum [Ca2+]i amplitude to ATP application immediately after a 10 sec nicotine administration 10 minutes after the control ATP application. Numbers in parenthesis indicate number of macrophages / number of preparations (equal to number of animals).
Mentions: The activation of macrophages by ATP and the inhibition of the ATP response by 100 µM nicotine was reliably recorded in each of the 21 preparations illustrated in Figure 2F. This allowed us to perform antagonists studies without the need to restudy the inhibitory response in those macrophages treated with the antagonists. Moreover, we thereby reduced the number of recording periods to a level that did not compromise signal strength and guaranteed reproducible ATP responses. In order to study the nAChR-subunits involved in the inhibitory effect of nicotine, we tested five different blockers with known subunit preferences [17] (Figure 3). The inhibitory effect of nicotine on ATP-evoked [Ca2+]i responses was unchanged in the presence of the non-selective ganglionic nAChR antagonist hexamethonium, the α3β4 nAChR-preferring antagonist mecamylamine or α7 nAChR-preferring antagonists α-bungarotoxin and methyllycaconitine (Figure 3A). However, the β2 nAChR-preferring antagonist di-hydro-β-eryhtroidine reversed the inhibitory effect of nicotine on ATP-evoked [Ca2+]i responses in macrophages (Figure 3B).

Bottom Line: 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.This inhibitory effect was reversed by the β2 nAChR preferring antagonist dihydro-β-eryhtroidine but not by hexamethonium (non-selective nAChR-antagonist), mecamylamine (α3β4 nAChR-preferring antagonist), α-bungarotoxin or methyllycaconitine (both α7 nAChR-preferring antagonist).

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Experimental Medicine, Translational Research Center for Gastrointestinal Disorders (TARGID), University of Leuven, Leuven, Belgium.

ABSTRACT

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 ([Ca(2+)]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 β2 and α7 nAChR was evaluated by β2 immunohistochemistry and fluorophore-tagged α-bungarotoxin.

Results: In 83% of macrophages cholinergic varicose nerve fibers were detected at distances <900 nm. The ATP induced [Ca(2+)]i increase was significantly inhibited in 65% or 55% of macrophages by 100 µM or 10 µM nicotine, respectively. This inhibitory effect was reversed by the β2 nAChR preferring antagonist dihydro-β-eryhtroidine but not by hexamethonium (non-selective nAChR-antagonist), mecamylamine (α3β4 nAChR-preferring antagonist), α-bungarotoxin or methyllycaconitine (both α7 nAChR-preferring antagonist). Macrophages in the stomach express β2 but not α7 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 β2 subunit of the nAChR is critically involved in the nicotine-induced inhibition of these resident macrophages.

Show MeSH