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A role for muscarinic receptors in neutrophil extracellular trap formation and levamisole-induced autoimmunity
Carmelo Carmona-Rivera, … , Mariana J. Kaplan, Peter C. Grayson
Carmelo Carmona-Rivera, … , Mariana J. Kaplan, Peter C. Grayson
Published February 9, 2017
Citation Information: JCI Insight. 2017;2(3):e89780. https://doi.org/10.1172/jci.insight.89780.
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Research Article Inflammation

A role for muscarinic receptors in neutrophil extracellular trap formation and levamisole-induced autoimmunity

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Abstract

Levamisole, an anthelmintic drug with cholinergic properties, has been implicated in cases of drug-induced vasculitis when added to cocaine for profit purposes. Neutrophil extracellular trap (NET) formation is a cell death mechanism characterized by extrusion of chromatin decorated with granule proteins. Aberrant NET formation and degradation have been implicated in idiopathic autoimmune diseases that share features with levamisole-induced autoimmunity as well as in drug-induced autoimmunity. This study’s objective was to determine how levamisole modulates neutrophil biology and its putative effects on the vasculature. Murine and human neutrophils exposed to levamisole demonstrated enhanced NET formation through engagement of muscarinic subtype 3 receptor. Levamisole-induced NETosis required activation of Akt and the RAF/MEK/ERK pathway, ROS induction through the nicotinamide adenine dinucleotide phosphate oxidase, and peptidylarginine deiminase activation. Sera from two cohorts of patients actively using levamisole-adulterated cocaine displayed autoantibodies against NET components. Cutaneous biopsy material obtained from individuals exposed to levamisole suggests that neutrophils produce NETs in areas of vasculitic inflammation and thrombosis. NETs generated by levamisole were toxic to endothelial cells and impaired endothelium-dependent vasorelaxation. Stimulation of muscarinic receptors on neutrophils by cholinergic agonists may contribute to the pathophysiology observed in drug-induced autoimmunity through the induction of inflammatory responses and neutrophil-induced vascular damage.

Authors

Carmelo Carmona-Rivera, Monica M. Purmalek, Erica Moore, Meryl Waldman, Peter J. Walter, H. Martin Garraffo, Karran A. Phillips, Kenzie L. Preston, Jonathan Graf, Mariana J. Kaplan, Peter C. Grayson

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Figure 4

Levamisole-induced NETs promote endothelial cell cytotoxicity and endothelial dysfunction.

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Levamisole-induced NETs promote endothelial cell cytotoxicity and endoth...
Human microvascular endothelial cells (HMVECs) were incubated in presence of levamisole (leva), spontaneously generated NETs (NETs), or levamisole-induced NETs (NETs-leva) for 24 hours. (A) Cytotoxicity assay was performed after 24 hours of incubation by colorimetric plate assay, as described in Methods. Results are the mean ± SEM of 6 independent experiments. For statistical analyses, Mann-Whitney U test was used; ***P < 0.001. (B) NETs generated by levamisole impair endothelium-dependent vasorelaxation in response to acetylcholine (Ach). Results are the mean ± SEM of 3 independent experiments. For statistical analyses, 2-way ANOVA with post-hoc Tukey’s test was used; **P < 0.01, ***P < 0.001.

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