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

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 3

Levamisole mediates NET formation through activation of M3.

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Levamisole mediates NET formation through activation of M3. (A) Quantifi...
(A) Quantification of NETs after stimulation with levamisole in the presence or absence of M1 inhibitors (Tele, VU) or M3 inhibitors (4-DAMP, DARIF) at concentrations mentioned in the Methods for 2 to 3 hours. Results are the mean ± SEM of 4 independent experiments. For statistical analyses, Kruskal-Wallis with post-hoc Dunn’s test was used; ****P < 0.0001. (B) Representative immunofluorescence images of nonpermeabilized neutrophils stimulated with levamisole in the presence or absence of M1 inhibitors (Tele, VU) or M3 inhibitors (4-DAMP, DARIF). (C) Quantification of NETs in wild-type (B6), muscarinic receptor 1 (M1) KO, M2 KO, or M3 KO mouse neutrophils after stimulation with levamisole or PMA for 4 hours. Results are the mean ± SEM of 4 independent experiments. For statistical analyses, Kruskal-Wallis with post-hoc Dunn’s test was used; *P < 0.05, ***P < 0.001. (D) Representative immunofluorescence images of nonpermeabilized mouse neutrophils incubated with levamisole or PMA for 4 hours. Scale bar: 20 μm.