Background/Purpose: The Vasculopathy induced by Cocaine-Levamisole (VICOL) is a recently described vasculopathy characterized by the presence of tissue necrosis. One of the possible mechanisms associated with this disease is the presence of NETosis a type of cell neutrophil cell death. The content of Nets is involved in endothelial activation and could explain the clinical manifestations. The muscarinic receptors M3 could be activated by levamisole and induce Netosis, an event that triggers the inflammation associated with the disease.

Methods: Neutrophils from healthy controls were isolated on a density gradient Ficoll-Dextran 3%, and cell viability and purity were assessed by stain with PI, DIOC6, and CD45-PCy7 and CD33-PE, respectively. After that, the induction of NETosis was evaluated through treatment with 40µM of Cocaine and 20nM of Levamisole and a mix of two components (40µM and 20nM). Then, neutrophils were cultured in polystyrene test tubes for 6 hours. As a positive control, 10 nM PMA was used. The presence of cell death was established by stains with 2µM Sytox and 1µg/µl Hoechst using cytometry. The double-positive cells indicate the presence of cell death. For the evaluation of inhibition, 10 nM and 20 nM of Hyoscine Butylbromide were added to the cultures before the stimulus, and the presence of cell death was also evaluated by cytometry. Flow cytometer data was analyzed using FlowJo, and statistical analysis was performed using GraphPad Prism V.8.

Results: The induction of neutrophil cell death was observed in the presence of cocaine and levamisole. A mean of 2.52% (range 0.443-3.35) and 20.28% (8.02-44.7) of neutrophils suffer cell death in the presence of cocaine and levamisole, respectively. The mix of Cocaine-Levamisole increases the median of cell death by 67.43% (31.3-88.8). The PMA was used as a control and induce cell death of 17.96% (11.3- 25.6). The cell death inhibition was assessed in neutrophils treated with Cocaine-Levamisole in the presence of 10nM and 20nM Hyoscine Butylbromide. The percentage of inhibition observed was 59.15% (15.9-80.3) with 10 nM of inhibitor and 40.95% (4.9-79.4) with 20 nM of inhibitor. As negative control not treated- neutrophils were evaluated and showed a cell death induction of 5,86% (3.5-8.9).

Conclusion: These results show that Levamisole induces a higher proportion of cell death than Cocaine, the mix of Cocaine-Levamisole enhanced the percentage of cell death. Hyoscine Butylbromide can inhibit the cell death induced by Cocaine-Levamisole. This is the first evidence of the capacity of Hyoscine Butylbromide to inhibit this phenomenon. In this study, we used flow cytometry as a tool for the evaluation of cell death that suggests NETosis. However, this strategy of flow cytometry should be assessed with antibodies against molecules involved in NETosis. The evidence of this study could suggest the potential use of this medicament to reduce the clinical manifestations of VICOL.

Carmona-Rivera C, et al. A role for muscarinic receptors in neutrophil extracellular trap formation and levamisole-induced autoimmunity. JCI Insight. 2017 Feb 9;2(3):e89780.

Sponsored by ASOREUMA and Programa de Sostenibilidad Universidad de Antioquia

Figure 1: Hyoscine Butylbromide inhibits neutrophil cell death induction by cocaine-levamisole. Percentage of neutrophils cell death stimulated by PMA [10 nM], cocaine (C) [40 µM], levamisole (L) [20 nM] cocaine-levamisole (C-L) and non-stimulated cells (n=5). Inhibition of cell death by Hyoscine Butyl bromide (BBH) at 10nM and 20 nM induced by cocaine-levamisole (C-L+I) (n=5). ANOVA I and Bonferroni comparison of means was used to calculate the p-value. Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/hyoscine-butylbromide-inhibits-neutrophil-cell-death-induction-by-cocaine-levamisole-a-proof-of-concept-for-the-management-of-vasculopathy-induced-by-cocaine-levamisole/