NETosis Secondary to the Use of Levamisole-Adulterated Cocaine: A Likely Underlying Mechanism of Vasculopathy

Abstract

Background: Since 2010, several cases of a new vasculopathy induced by the use of levamisole-adulterated cocaine (LAC) have been reported. This vasculopathy is characterized by retiform purpura, earlobe necrosis, multisystem compromise, and multiple autoantibodies. Given its similarity to antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, LAC-associated vasculopathy is postulated to be mediated by pathophysiologic processes resulting from neutrophil cell death by NETosis, a phenomenon previously described in ANCA vasculitis. This study tries to establish the presence of NETosis induced by cocaine, levamisole, or both. Methodology. Neutrophils were isolated from the peripheral blood of healthy controls by Ficoll-Hystopaque density gradient centrifugation followed by dextran sedimentation. Cell viability and purity were evaluated by flow cytometry after staining with PI/DiOC6 and labeling with fluorescent anti-CD45/anti-CD3 monoclonal antibodies (mAbs), respectively. Neutrophils were exposed to levamisole, cocaine, a cocaine-levamisole mixture, and sera pools from healthy controls and patients with LAC-associated vasculopathy. NETosis was then assessed by flow cytometry after staining cells with Sytox Green, Hoechst-33342, and fluorescent antineutrophil elastase (NE) and antimyeloperoxidase (MPO) mAbs. In addition, NETosis was morphologically confirmed by fluorescence microscopy. Proinflammatory cytokine levels in culture supernatants and reactive oxygen species (ROS) synthesis were determined by flow cytometry. The involvement of calcium and muscarinic receptors in cell death induction was evaluated in parallel experiments carried out in the presence of 1,2-bis (o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid (BAPTA) and hyoscine butylbromide (HBB), their respective inhibitors.

Results: Cocaine, levamisole, and a cocaine-levamisole mixture induced neutrophil cell death. DNA/MPO extrusion and cell morphology patterns were consistent with NETosis. Neither proinflammatory cytokines nor ROS behaved as proNETotic factors. Preliminary results suggested that muscarinic receptors and calcium-dependent signals were involved in LAC-induced NETosis.

Conclusions: Cocaine, levamisole, and a cocaine-levamisole mixture can induce NETosis through mechanisms involving muscarinic receptors and calcium-dependent pathways.

Figure 1

Purity and viability of neutrophil-enriched suspensions. Circulating neutrophils from healthy controls were isolated by Ficoll-Hystopaque density gradient centrifugation followed by dextran sedimentation. Cell purity and viability were evaluated by labeling with anti-CD45-PE/Cy7 and anti-CD33-PE mAbs and PI/DiOC₆ staining, respectively. (a) Representative dot plots showing the strategy for gating CD44⁺CD33⁺ neutrophils and evaluating cell purity. (b) Representative dot plot illustrating the distinction of live cells (DiOC₆^bright IP^neg), cells with mitochondrial injury (DIOC₆^dimIP^neg), and cells with compromised plasma membrane (DIOC₆^negIP⁺⁺⁺). n = 4 independent experiments.

Figure 2

Induction of NETosis in neutrophil cultures exposed to cocaine, levamisole, and cocaine-levamisole mixture. Neutrophil-enriched suspensions from healthy controls were exposed to cocaine (40 µM), levamisole (20 nM), cocaine-levamisole mixture (20 µM/40 nM), and PMA (20 nM) for 6 h at 37°C. Staining with Sytox Green, Hoechst 33342, and mAbs anti-MPO-PE and anti-NE-Alexa fluor 647 was used to evaluate extrusion of neutrophil DNA and granular content. (a) Representative dot plots illustrating the strategy for gating double positive Sytox Green⁺ Hoechst 33342⁺ netting neutrophils. (b-c) Representative dot plots and column chart showing the percentage of netting neutrophils in response to each treatment. Bar represents median values and error bars correspond to IQRs. ^∗p ≤ 0.05; Kruskal–Wallis test. n = 4 independent experiments. (d) Immunofluorescence analysis by confocal microscopy confirming NET formation. Magnification: 200x. n = 3 independent experiments.

Figure 3

Evaluation of MPO in NETs extruded from cultures of neutrophils exposed to cocaine, levamisole, and cocaine-levamisole mixture. Neutrophil-enriched suspensions from healthy controls were exposed to cocaine (20 µM), levamisole (40 nM), cocaine-levamisole mixture (20 µM/40 nM), and PMA (20 nM) for 6 h. Staining with Sytox Green, Hoechst 33342, and anti-MPO-PE and anti-NE-Alexa fluor 647 mAbs was used to evaluate extrusion of neutrophil DNA and granular content. (a) Representative dot plots and (b) column chart showing the percentages of double positive Hoechst 33342⁺ MPO⁺ netting neutrophils generated in response to each treatment. Bar represents median values and error bars correspond to the IQRs. ^∗p ≤ 0.05; Kruskal–Wallis test. n = 4 independent experiments.

Figure 4

Induction of NETosis in neutrophil cultures exposed to sera from patients with LAC-associated vasculitis. Neutrophil-enriched suspensions from healthy controls were exposed to pooled sera (20%) from healthy controls (PHCs) and patients with LAC-associated vasculitis with GN (PWN) or without GN (PWON) for 6 h at 37°C. Staining with Sytox Green, Hoechst 33342, and anti-MPO-PE and anti-NE-Alexa fluor 647 mAbs was used to evaluate extrusion of neutrophil DNA and granular MPO. (a-b) Representative dot plots and column chart showing the percentages of double positive Sytox Green⁺ Hoechst 33342⁺ netting neutrophils generated in response to each treatment. (c-d) Representative dot plots and column chart showing the percentages of double positive MPO ⁺ Hoechst 33342⁺ netting neutrophils generated in response to each treatment. Bar represents median values and error bars correspond to IQRs. ^∗p ≤ 0.05; Kruskal–Wallis test. n = 4 independent experiments. (e) Immunofluorescence analysis by confocal microscopy confirming NET formation. Magnification: 200x. n = 4 independent experiments.

Figure 5

IL-8 levels produced by neutrophil cultures exposed to cocaine, levamisole, and cocaine-levamisole mixture. Supernatants collected from cultures described in Figures 3 and 4 were kept a −80°C. Then, they were thawed to quantify the levels of proinflammatory cytokines. Only IL-8 showed changes. (a) Cocaine, levamisole, and the cocaine-levamisole mixture and (b) pooled sera from healthy controls (PHCs) and patients with LAC-associated vasculitis with (PWN) or without (PWON) GN. The average basal concentration of IL-8 in PHC (violet line), PWON (red line), and PWN (blue line) are shown with their corresponding values. Graphs show median values and IQRs. ^∗p ≤ 0.05; Kruskal–Wallis test. n = 1 independent experiments.

Figure 6

ROS generation in neutrophil cultures exposed to cocaine, levamisole, and cocaine-levamisole mixture. Neutrophil-enriched suspensions were incubated with dihydrorhodamine 123 (1 : 1000) for 10 min before adding cocaine (20 µM), levamisole (40 nM), a cocaine-levamisole mixture (20 µM/40 nM), and PMA (20 nM) for 20 and 60 min, washed, and analyzed by flow cytometer. (a-b) Representative histograms and (c) column chart showing MFI of rhodamine 123 in neutrophils under each treatment. Graphs show median values and error bars correspond to IQRs. ^∗p ≤ 0.05; Kruskal–Wallis test. n = 3 independent experiments.

Figure 7

Role of intracellular calcium in NETosis induced by a cocaine-levamisole mixture. Neutrophil-enriched suspensions from healthy controls were incubated with BAPTA at different concentrations for 10 min before adding levamisole (40 nM) and the cocaine-levamisole mixture (20 µM/40 nM) for 3 h. After incubation, cells were stained with Sytox Green and Hoechst 33342, washed, and analyzed by flow cytometry. (a) Representative dot plots showing double positive Sytox Green⁺ Hoechst 33342⁺ netting neutrophils in presence of BAPTA. (b) Representative dot plots showing the effect of 2 µM BAPTA on reduction of double positive Sytox Green⁺ Hoechst 33342⁺ netting neutrophils induced in the presence of the cocaine-levamisole mixture or levamisole. n = 2 independent experiments.

Figure 8

Role of muscarinic receptors in NETosis induced by a cocaine-levamisole mixture. Neutrophil-enriched suspensions were incubated with 20 nM HBB for 10 min and then exposed to cocaine (20 μM), levamisole (40 nM), and the cocaine-levamisole mixture (20 µM/40 nM) and PMA (20 nM) for 6 h at 37°C. After incubation, cells were stained with Sytox Green and Hoechst 33342, washed, and analyzed by flow cytometry. (a-b) Representative dot plots and column chart showing the percentages of double positive Sytox Green⁺ Hoechst 33342⁺ netting neutrophils induced by each treatment. Bars show median values and error bars correspond to IQRs. ^∗p ≤ 0.05; Kruskal–Wallis test. n = 4 independent experiments. (c) Immunofluorescence analysis by confocal microscopy confirming the inhibition of NET formation by HBB. Magnification: 200x. n = 4 independent experiments.

Figure 9

Role of muscarinic receptors in NETosis induced by sera from patients with LAC-associated vasculitis. Neutrophil-enriched suspensions from healthy controls were incubated with HBB (20 nM) for 10 min and then exposed to pooled sera (20%) from healthy controls (PHCs) and patients with LAC-associated vasculitis with (PWN) or without (PWON) GN for 6 h at 37°C. After incubation, cells were stained with Sytox Green and Hoechst 33342, washed, and analyzed by flow cytometry. (a) Representative dot plots and (b) column chart showing the percentage of double positive Sytox Green⁺ Hoechst 33342⁺ netting neutrophils generated in response to each treatment. Bars represent median values and error bars correspond to IQRs. ^∗p ≤ 0.05; Kruskal–Wallis test. n = 4 independent experiments. (c) Representative images from confocal microscopy showing Sytox Green⁺ Hoechst 33342⁺ netting neutrophils. Magnification: 200x. n = 4 independent experiments.

Figure 10

A model of the immunopathogenic mechanism underlying LAC-associated vasculitis.