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

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.


Introduction
Vasculopathies are diseases of the blood vessels that can cause ischemic lesions in diferent organs, ranging in severity from mild to severe enough to cause death [1].Infammation of blood vessels is a type of vasculopathy and comprises several diseases known as vasculitis.Vasculitis are classifed according to the caliber of the afected vessels into large artery, medium vessel, and small vessel vasculitis or according to their etiology into primary and secondary vasculitis.Primary vasculitis are those in which no specifc triggering stimulus can be identifed, while secondary vasculitis are induced by drugs, infections, neoplasms, or autoimmune diseases [2,3].
Levamisole is an anthelmintic drug with nonspecifc immunomodulatory properties.However, in 1999, it was withdrawn from the US market and restricted to veterinary use because its administration was associated with purpuric lesions on both cheeks and necrosis of earlobes [4,5].In 2002, the use of levamisole as a cocaine additive was frst reported in the US.Such adulteration increased from 1% to 70% in less than a decade [4,6,7].In 2010, several cases of levamisole-adulterated cocaine (LAC)-induced vasculopathy were described.Patients exhibited a variety of clinical manifestations, including painful retiform purpura with or without central necrosis and hemorrhagic blisters, as well as elevated titers of antinuclear autoantibodies (ANAs) and antineutrophil cytoplasmic autoantibodies (ANCAs), including those directed against granule myeloperoxidase (MPO) and proteinase 3 (PR3) [8,9].
An important group of primary vasculitis comprises ANCA-associated vasculitis (AAVs) [10,11] characterized by circulating ANCAs directed against MPO and PR3 proteins of neutrophil granules.Neutrophil cell death is implicated in the pathophysiology of AAVs.However, it is essential to note that neutrophils can undergo necrosis, apoptosis, and NETosis.NETosis is a type of death characterized by neutrophil extracellular trap (NET) formation resulting from the extrusion of chromatin and granule proteins.Tis type of death appears to play a crucial role in the pathogenesis of vasculitis.
Levamisole is known to induce the release of NETs [12].However, the efect of the cocaine-levamisole combination on NETosis and its implications for the pathophysiology and clinical presentation of LAC-associated vasculitis is unknown.
NETosis can be induced by diferent mechanisms such as the activation of muscarinic receptors, the production of ROS-catalyzed by the NADPH-oxidase complex-or the increase in intracellular calcium levels, which in turn activates the enzyme peptidyl-arginine deiminase 4 (PAD4) [13].Although some studies have described the underlying mechanism of levamisole-induced NETosis, the mechanism by which the cocaine-levamisole combination could induce NETosis is still unknown.
Understanding the underlying mechanisms of LACassociated vasculitis could provide therapeutic options, as current treatment is based on immunosuppressants, which are related to various adverse events.For example, as levamisole activates muscarinic receptors expressed on neutrophils, treatments could be designed to block muscarinic receptor-dependent signaling by inhibitors such as hyoscine butylbromide (HBB).

Patients and Controls.
Tis study included patients with a confrmed diagnosis of LAC-associated vasculitis (n � 25) who were evaluated by the Rheumatology Group of the University of Antioquia (GRUA, for its acronym in Spanish) at the Rheumatology Service of the Hospital Universitario San Vicente Fundación in Medellín.Patients were classifed into two groups, those with glomerulonephritis (GN) and those without GN, according to their clinical information.Healthy individuals matched by sex and similar in age to the group of patients were also included as controls (n � 10).All individuals were of legal age, agreed to participate in the study voluntarily, and signed the informed consent form.Exclusion criteria were active infection at blood sampling, diagnosis of malignant neoplasia, and the impossibility of accessing the clinical or paraclinical records necessary for the analyses proposed in the study.

Preparation of Pooled
Sera.Peripheral blood samples (6 mL) were drawn from patients and healthy controls.Blood samples were allowed to clot at room temperature (RT) and then centrifuged at 900 × g 10 min at RT to separate the serum.Serum samples were aliquoted and stored at −20 °C until use.Pooled sera were prepared from samples from healthy controls, patients with GN, and patients without GN.

Isolation of Neutrophils.
Peripheral blood samples (8 mL) were drawn from healthy controls in tubes containing acid citrate dextrose (ACD) anticoagulant.Te blood sample was brought to a fnal volume of 10 mL with 1X PBS.Tis mixture was gently added to the walls of a 15 mL tube containing 3 mL of Hystopaque-1077 and centrifuged at 900 × g for 30 min at RT. Ten, the top three layers (plasma, mononuclear cell, and isolation media) were removed.Te bottom layer containing erythrocytes and neutrophils was suspended in PBS to a volume of 7 mL, mixed with an equal volume of 3% dextran, and incubated at RT for 20 min.Te neutrophil population was then carefully removed and transferred to a 15 mL tube, resuspended in PBS to complete 14 mL, and centrifuged at 650 × g for 7 min.Ten, the 2 Journal of Toxicology supernatant was discarded, and cells were suspended in 5 mL of hypotonic PBS, allowed to stand for 30 s, mixed with 5 mL of re-equilibration PBS, and centrifuged at 650 × g for 7 min.Tis step was repeated once more.Finally, cells were resuspended in 600 µL of RPMI without SBF, and their count was determined in a Neubauer chamber using the trypan blue dye exclusion method.Ten, 100,000 cells were suspended in 200 µL of RPMI to determine their viability and purity in BD LSRFortessa ™ (BD Biosciences °C and acquired on a BD LSRFortessa ™ (BD Biosciences).Data were analyzed using the FlowJo software.According to Zharkova et al. [14], netting neutrophils were evidenced as double-positive Hoechst 33342 + and Sytox Green + events.Tese two dyes stain nucleic acids but difer because Hoechst 33342 crosses the cell membrane and Sytox Green does not, so it will only bind to nucleic acids when the cell membrane is damaged and the genetic material is exposed (Figure 2(a)).(20 nM), as a positive control of ROS generation, for 20 min and 60 min.After incubation, cells were placed on ice for 10 min to stop the reaction and washed twice with 1X PBS and centrifugation at 450 × g for 5 min at 4 °C.Subsequently, supernatant was removed, and cells were resuspended in PBS and kept on ice until acquisition on the BD LSRFortessa ™ (BD Biosciences).Data were analyzed using the FlowJo software.

Statistics.
Demographic and clinical characteristics of patients and healthy controls are described as medians and percentages.Results from diferent treatments were compared with the Kruskall-Wallis test followed by Dunn's test for multiple comparisons.Biological replicates (n � 2-8) of each assay were independently carried out and are indicated in the corresponding fgures.Data are shown in column charts (medians and interquartile ranges).Diferences were considered signifcant when p < 0.05.Statistical analyses were run in the GraphPad Prism 8.0.1 software.

Induction of NETosis in Neutrophil Cultures Exposed to Pooled Sera from Patients with LAC-Associated Vasculitis.
To evaluate whether soluble serum component from patients with LAC-associated vasculitis could induced NETosis, neutrophil-enriched cell suspensions from healthy controls were incubated with PHC, PWN, and PWON pooled sera.Similar percentages of double positive Sytox Green + Hoechst-33342 + cells were generated in the presence of PWN (median � 10.6% and IQR: 2.39-14.3)and PWON (median � 11.4% and IQR: 5.97-15.00).Tese values were lower than those generated by the cocaine-levamisole mixture but higher than those from cultures incubated with PHC (median � 5.1% and IQR: 3.35-8.75)(Figure 4).In contrast, the IL-8 levels increased in neutrophil cultures exposed to diferent sources of serum PHC (mean basal and stimulated: 1,84-427 pg/mL), PWON (mean basal and stimulated: 458-508 pg/mL), and PWN (mean basal and stimulated: 1,52-399 pg/mL), but no signifcant diference was observed between the three serum sources (Figure 5).

ROS Production in Neutrophil Cultures
Exposed to Cocaine, Levamisole, and Cocaine-Levamisole Mixture.To assess whether cocaine and levamisole treatment induced the formation of NETs through the release of ROS, neutrophils were incubated with cocaine, levamisole, and a cocainelevamisole mixture for 20 and 60 min.Afterwards, DHR123 was added, and its conversion to rhodamine mediated by superoxide anion was assessed by fow cytometry.Cocaine induced a slight increase (1.3x) in ROS production.Interestingly, cultures treated with levamisole showed lower ROS synthesis than the negative control.Moreover, ROS production was lowered in cultures exposed to the cocaine- levamisole mixture than to cocaine (Figure 6), suggesting an antagonistic efect of levamisole on cocaine-induced ROS production and that it might not be involved in the induction of NETosis (Figure 6).Tis enzyme participates in chromatin decondensation and subsequent NET extrusion from neutrophils.To determine whether the NETosis induced by the cocaine-levamisole mixture was due to changes in the intracellular calcium level, neutrophils were preincubated with BAPTA (2 μM and 20 μM), a calcium chelator, for 3 h before adding cocaine, levamisole, and a cocaine-levamisole mixture.Te percentage of netting neutrophils was 40% in cultures treated with the cocaine-levamisole mixture and decreased to MFI 21% in the presence of BAPTA (Figure 7).Tese results suggest that cocaine and levamisole combined could induce NETosis by regulating intracellular calcium levels; however, further assays are needed to confrm this hypothesis.

Role of Muscarinic Receptors in NETosis
Induced by a Cocaine-Levamisole Mixture.Hyoscine butylbromide is a quaternary ammonium compound with an anticholinergic efect.It has an antagonistic efect on muscarinic receptors present in diferent cells, including neutrophils [16].To determine whether NETosis induced by cocaine, levamisole, and the cocaine-levamisole mixture involved muscarinic receptors, neutrophils were incubated with 20 µL of HBB (20 nM) for 10 min before exposure to cocaine, levamisole, and a cocaine-levamisole mixture for 6 h at 37 °C.After incubation, cells were stained with Sytox Green and Hoechst 33342, washed, and analyzed by fow cytometry.BBH reduced the frequency of double positive Sytox Green + Hoechst + netting neutrophils by MFI 18.9%.Tis reduction was confrmed by fuorescence microscopy (Figures 8(a)-8(c)).

Role of Muscarinic Receptors in NETosis Induced by Sera from Patients with LAC-Associated Vasculitis.
To determine whether muscarinic receptors were also involved in NETosis induced by sera from patients with LAC-associated vasculitis, neutrophils were incubated with 20 µL of 20 nM BBH for 10 min before exposure to PHC, PWN, and PWON pooled sera for 6 h at 37 °C.After incubation, cells were stained with Sytox Green and Hoechst 33342, washed, and analyzed by fow cytometry.Preincubation with HBB did not change the percentages of netting neutrophils in cultures exposed to PWN (9.5% ± 1.12%-9.3%± 0.89%) or PWON (11.02% ± 0.85%-8.6%± 0.93%).No changes were observed in either of the cultures exposed to the PHC (5.6% ± 0.56%-3.8%± 0.42%) (Figure 9).

Discussion
In the present work, neutrophils from healthy individuals exposed in vitro to cocaine, levamisole, and a cocainelevamisole mixture released NETs containing MPO but not EN.Carmona et al. had reported levamisole-induced NETosis [13] and Lood and Hunges had demonstrated the release of NE-enriched NETs in response to cocaine or levamisole [17].MPO release is a crucial event for NET formation.Indeed, Metzler et al. reported an increased risk of infection in MPO-immunodefcient individuals related to their inability to release NETs [18].Te presence of MPO but not EN observed in NETs released from neutrophils exposed to the cocaine-levamisole mixture suggests that this combination could induce NETosis through a diferent mechanism.Moreover, it would explain the predominance (92%) of circulating ANCAs and anti-MPO antibodies in the group of patients with LAC-associated vasculitis.
Te cocaine-levamisole combination presented a synergistic efect on NETosis induction.Tis efect could be mediated by a levamisole-associated neuropharmacological mechanism that inhibits the enzyme monoamine oxidase and activates receptors, thus increasing the transmission of dopamine signals and activation of NETosisassociated mechanisms [19,20].Another hypothesis attributes the synergistic efect of the cocaine-levamisole mixture to aminorex, a metabolite of levamisole that enhances the 10 Journal of Toxicology stimulant properties of cocaine.Prolonged consumption of aminorex has been associated with pulmonary hypertension and vasculitis [21,22].Infammation depends on immunopathogenic and nonimmunopathogenic mechanisms.In the case of vasculitis, immunopathogenic mechanisms comprise those mediated by cells of the immune system, such as cytotoxic T cells directed against blood vessel components, the expression of adhesion molecules on endothelial cells, and the presence of antibodies that can form immune complexes that directly damage endothelial cells or are deposited on blood vessel walls [23].In the present work, patients with LAC-associated vasculitis, confrmed by skin biopsy, were positive for ANCAs (92%), anti-MPO antibodies (64%), or anti-PR3 antibodies (28%).Because some patients (36%) had immune complex (IC)-mediated nephritis, it was hypothesized that ICs might cause the neutrophil cell death that characterizes LAC-associated vasculitis.Terefore, to determine whether ICs could induce NETosis, neutrophils were incubated with pooled sera from patients with nephritis (higher likelihood of circulating ICs) or patients without renal involvement (lower likelihood of circulating ICs).[13].In the present study, HBB did not inhibit the proNETotic efect of PMA but inhibited partially that of the cocaine-levamisole mixture, suggesting that the RAF/MEK/ ERK pathway may be not involved in the induction of NETosis by cocaine and levamisole combined.Tese results support the administration of HBB as a therapeutic option for patients with LAC-associated vasculitis, as partial inhibition of NETosis could reduce clinical manifestations and limit the use of immunosuppressants and their associated adverse efects.
Several mechanisms of NETosis induction have been described, such as the activation of NADPH oxidase and the increased intracellular calcium levels.Activation of NADPH oxidase results in the synthesis of ROS that promotes the release of proteins from neutrophil cytoplasmic granules.Te increase in intracellular calcium levels activates the peptidylarginin deiminase 4 (PAD4), an enzyme involved in histone citrullination.According to the results of the present work, cocaine, but not levamisole, induced ROS synthesis by neutrophils after 60 min of incubation.Moreover, the cocaine-levamisole mixture reduced ROS levels, suggesting an antagonistic efect of levamisole on cocaine-induced ROS generation.In contrast, Carmona et al. observed that levamisole induces the generation of ROS through the activation of the NADPH oxidase but not mitochondrial synthesis of the oxygen radicals, event that could be explained by the fndings of [13].
As mentioned above, NETosis can be induced by an elevated intracellular calcium level that activates the PAD4 enzyme [25].In the present study, BAPTA-an aminocarboxylic acid that chelates two calcium ions and thereby decreases free cytoplasmic calcium level-inhibited the NETosis induced by the cocaine-levamisole mixture.Tese preliminary results suggested that muscarinic receptors on the plasma membrane of neutrophils could mediate the induction of NETosis by the cocaine-levamisole mixture.Such receptors induce the activation of tyrosine kinases that phosphorylate phospholipase C. Tis enzyme hydrolyzes phosphatidylinositol bisphosphate to diacylglycerol and inositol triphosphate (IP3).Te IP3 then binds to receptors in the endoplasmic reticulum to induce calcium release [26].
Elevated intracellular calcium levels in neutrophils activate the enzyme PAD4, which participates in chromatin decondensation, a hallmark of NETosis.Carmona et al. demonstrated that CI-amidine, a PAD4 inhibitor, decreased the percentage of netting neutrophil cells in cultures treated with levamisole [13].In a murine model, Hemmers et al. observed that, in contrast to control mice, PAD4-defcient mice did not produce NETs after exposure to the infuenza A virus.Tese results further supported that PAD4 is required to trigger neutrophil cell death by NETosis [27].According to some studies, the citrullination of histones H3 and H4 is required for NET formation [28][29][30].
In addition, we evaluated whether proinfammatory cytokines could play a role in NETosis induced by the cocaine-levamisole mixture.Terefore, levels of IL-1β, IL-6, TNF-α, IL12-P70, and IL-8 were quantifed in the supernatants of cultures exposed to each compound and the cocaine-levamisole mixture.Neutrophils only synthesized IL-8 in response to these treatments, in agreement with a study by Altstaedt et al. on the constitutive induction of IL-8 synthesis in neutrophils [31].Likewise, neutrophils exposed to a pooled serum from patients with LAC-associated vasculitis showed a tendency to higher levels of IL-8 concentrations; these levels are the product of basal IL-8 concentrations and the IL-8 induced by serum.
Sera from patients with LAC-associated vasculitis can induce IL-8 synthesis, which may be due to their anti-MPO content.In Hsieh et al.'s study, neutrophils exposed to ANCA-MPO and ANCA-PR3 from ANCA vasculitis patients increased IL-8 levels in the presence of ANCA-MPO; these fndings suggest that this antibody could be an inducer of IL-8 production [32].
In summary, cocaine and levamisole can induce NETosis through diferent pathways as follows: (i) activation of muscarinic receptors on the neutrophil cell membrane.Tis pathway is followed by the release of cytoplasmic granular proteins that translocate to the nucleus to decondense chromatin and generate NETs enriched in nuclear material and granular enzymes such as MPO.(ii) Increasing levels of intracellular calcium.Upon activation, IP3 generation would promote calcium release from the endoplasmic reticulum.In turn, intracellular calcium would activate PAD4 to mediate chromatin decondensation and subsequent release of NETs.
Te muscarinic inhibitor HBB partially decreased NETosis induced by the cocaine-levamisole mixture, thus evidencing the involvement of muscarinic receptors in the induction of this form of cell death.Moreover, muscarinic receptors can also activate the intracellular calcium mobilization.
Finally, we propose a model to relate NETosis induction by the cocaine-levamisole mixture to the clinical manifestations of LAC-associated vasculitis (Figure 10).According to this model, antigen-presenting cells would recognize the MPO on NETs and present it to CD4 + T-cells.Te activated T-cells would promote the activation of B-cells, leading to the synthesis of autoantibodies directed against MPO (ANCA-MPO).Tese autoantibodies, in turn, would amplify other cellular mechanisms such as the activation of more neutrophils through Fc receptors, the release of NETs, activation of endothelial cells to express adhesion molecules, decrease of blood fow, and binding of neutrophils to the endothelium.Tis way, an infammatory and thrombotic environment would ensue and cause the characteristic clinical manifestations of LAC-associated vasculitis, such as necrosis of earlobes.

Conclusions
Cocaine and levamisole act synergistically to induce neutrophil cell death characterized by DNA and MPO extrusion.Although extracellular NE was not detected in neutrophils exposed to the cocaine-levamisole mixture, the detection of extracellular DNA and MPO, and the cellular characteristics evidenced by fuorescence microscopy, suggest that the combination of the two molecules induced neutrophil cell death by NETosis.
Sera from patients with LAC-associated vasculitis also induced DNA and MPO release from neutrophils with a pattern morphologically compatible with NETosis although to a lesser degree than the cocaine-levamisole mixture.
Te mechanism underlying the NETosis induced by the cocaine-levamisole mixture seems to be independent of ROS generation.Levamisole did not induce ROS synthesis and even antagonized that induced by cocaine.Preliminary results suggest that NETosis induced by the cocaine-levamisole mixture depends on a calcium-dependent pathway.
HBB, a muscarinic receptor inhibitor, partially reduced the simultaneous efect of cocaine and levamisole on neutrophils, providing evidence for the involvement of these receptors in the induction of NETosis when both compounds are combined.
Finally, the present results propose fow cytometry as a valuable tool for the study of NETosis, given its simplicity, reproducibility, and low probability of data loss, as well as for being a more agile and higher throughput process.

Figure 1 :
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/DiOC 6 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 (DiOC 6 bright IP neg ), cells with mitochondrial injury (DIOC 6 dim IP neg ), and cells with compromised plasma membrane (DIOC 6 neg IP +++ ).n � 4 independent experiments.

Figure 2 :
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 fuor 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) Immunofuorescence analysis by confocal microscopy confrming NET formation.Magnifcation: 200x.n � 3 independent experiments.

Figure 3 :
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 fuor 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 :
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 fuor 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) Immunofuorescence analysis by confocal microscopy confrming NET formation.Magnifcation: 200x.n � 4 independent experiments.
Induced by a Cocaine-Levamisole Mixture.Te increase in intracellular calcium level is one of the mechanisms involved in the induction of NETosis since it activates the enzyme PAD4.

Figure 5 :Figure 6 :
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.Ten, they were thawed to quantify the levels of proinfammatory 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.Te 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 7 :
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 diferent 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 fow cytometry.(a) Representative dot plots showing double positive Sytox Green + Hoechst 33342 + netting neutrophils in presence of BAPTA.(b) Representative dot plots showing the efect 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 :
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 fow 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) Immunofuorescence analysis by confocal microscopy confrming the inhibition of NET formation by HBB.Magnifcation: 200x.n � 4 independent experiments.

Figure 9 :
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 fow 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.Magnifcation: 200x.n � 4 independent experiments.

Figure 10 :
Figure 10: A model of the immunopathogenic mechanism underlying LAC-associated vasculitis.

Table 1 :
Sociodemographic and clinical characteristics of patients with LAC-associated vasculitis (n � 25).