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.

Conflict of interest statement

The authors have declared that no conflict of interest exists.

Figures

Figure 1. Levamisole induces NET formation in a nicotinamide adenine dinucleotide phosphate oxidase– (NOX-) and peptidylarginine deiminase–dependent (PAD-dependent) manner.

Neutrophils from healthy controls were incubated with 100 μM levamisole or 2.5 μM calcium ionophore A23187 (Io) for 3 hours. (A) Sytox green assay was used to quantify neutrophil extracellular DNA release after stimulation. Results are the mean ± SEM of 4 independent experiments. For statistical analyses, Kruskal-Wallis with post-hoc Dunn’s test was used. (B) Immunofluorescence analysis quantifies NET formation after 3 hours of incubation with levamisole (neutrophil elastase [red] and DNA [blue]). Scale bar: 20 μm. (C) Nuclear envelope marker lamin B (red) depicts areas of neutrophil nuclear membrane disintegration after 3 hours of incubation with levamisole. Scale bar: 10 μm. (D) Neutrophil elastase (red) translocates from the cytoplasm into the nucleus after 3 hours incubation with levamisole. Scale bar: 10 μm. (E and F) Treatment with (E) 5 μM diphenylene iodonium (DPI; NOX inhibitor) or (F) 200 μM Cl-amidine (PAD inhibitor) significantly decreases the percentage of netting neutrophils in response to levamisole. Results are the mean ± SEM of 4 independent experiments. For statistical analyses, Mann-Whitney U test was used; *P <0.05, **P < 0.01. Scale bar: 20 μm.

Figure 2. Levamisole and acetylcholine induce NETs through Akt and ERK pathways.

(A) Quantification of M1 and M3 expression by flow cytometry in neutrophils from nonstimulated healthy controls (n = 5) and AAV (Vas) (n = 5) and SLE subjects (n = 5). Results are the mean ± SEM of 5 independent experiments. For statistical analyses, Kruskal-Wallis with post-hoc Dunn’s test was used; *P < 0.05. (B) Quantification of neutrophil DNA released following 3-hour stimulation with levamisole (leva) or acetylcholine (Ach) in presence or absence of the muscarinic receptor inhibitor atropine. Results are the mean ± SEM of 4 independent experiments. For statistical analyses, Mann-Whitney U test was used; *P < 0.05, **P < 0.01,***P < 0.001,****P < 0.0001. (C) Phosphorylation status of Akt and ERK1/2 assessed by Western blot in neutrophils from healthy controls stimulated with acetylcholine or levamisole for 30 minutes. Representative Western blot image from 2 independent experiments. (D) Quantification of neutrophil DNA released in presence or absence of PMA or levamisole in the presence or absence of an Akt inhibitor for 3 to 4 hours. Results are the mean ± SEM of 4 independent experiments. For statistical analyses, Mann-Whitney U test was used; **P < 0.01,****P < 0.0001.

Figure 3. Levamisole mediates NET formation through activation of M3.

(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.

Figure 4. Levamisole-induced NETs promote endothelial cell cytotoxicity and endothelial dysfunction.

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.

Figure 5. Autoantibodies to NET proteins are present in sera of users of levamisole-tainted cocaine.

Quantification of anti-LL37, anti-elastase, anti-histone H3, anti-citrullinated histone H3, anti-histone H4, and anti-citrullinated (cit) histone H4 in RA (n = 8), NIDA (asymptomatic active users of levamisole-tainted cocaine, n = 52), UCSF (levamisole-induced vasculitis, n = 6), or healthy donor (n = 20) sera. Results are expressed as OD index (OD in patient serum/mean OD in control sera). Results are displayed as the mean ± SEM. For statistical analyses, Kruskal-Wallis with post-hoc Dunn’s test was used; *P < 0.05.

Figure 6. NETs are present in skin lesions from patients with levamisole-induced vasculitis.

Formalin-fixed, paraffin-embedded cutaneous biopsy material obtained from 5 patients with confirmed levamisole-induced autoimmunity was stained for citrullinated-histone H4 (cit-H4), myeloperoxidase (MPO), or H&E. Representative images are displayed. Biopsy from patient 2572A demonstrated leukocytoclastic vasculitis in the small capillaries with karyorrhexis and intravascular thrombi with dense neutrophilic infiltrate in the deeper dermis. Biopsy from patient 8050A demonstrated perivascular and perieccrine neutrophilic infiltrates with areas of intravascular thrombosis. Biopsy from patient 8625A demonstrated a large, organized thrombus within a deep dermal artery. NETs (pink) were observed in associated areas of neutrophilic infiltrate in each of these specimens (arrows). Original magnification, ×40.