Session Type: ACR Concurrent Abstract Session
Session Time: 4:30PM-6:00PM
Levamisole, an anti-helminth drug, has been implicated in cases of drug-induced autoimmunity in humans exposed to adulterated cocaine. Clinical manifestations of levamisole-induced autoimmunity include cutaneous vasculitis and specific autoantibodies (e.g. anti neutrophil cytoplasmic antibodies, ANCA; antinuclear antibodies, ANA). Neutrophil extracellular trap (NET) formation is a novel cell death mechanism implicated in certain idiopathic autoimmune diseases. Proteins commonly found within NETs include the specific targets of ANCA and ANA. The objective of this study was to determine the role of NET formation in the pathogenesis of levamisole-induced autoimmunity.
Methods: Human and mouse neutrophils were exposed to graded concentrations of levamisole. NET formation was visualized and quantified by fluorescence microscopy. To assess the pathways involved in levamisole-induced NETosis, in some experiments the following inhibitors were added diphenylene iodonium (DPI), an inhibitor of reactive oxygen species, and Cl-amidine, an inhibitor of peptidylarginine deiminase (PAD) enzymes. Involvement of Akt and Raf-MEK-ERK kinase pathways was tested by Western blot. The role of Toll-like receptors (TLRs) and muscarinic receptors (MR) in levamisole-induced NETosis was investigated. Pharmacologic inhibitors and MR knockout mice were used to study specific MR subtypes implicated in NETosis. MR subtype receptors on neutrophils from patient samples were assessed by flow cytometry. Autoantibodies against specific NET-components were examined among cocaine users with no overt sign of autoimmune disease.
Results: Levamisole induced prominent NETs in human and murine neutrophils that contained myeloperoxidase and proteinase 3, the targets of ANCA. NET formation induced by levamisole was dependent on phosphorylation of Akt and Raf-MEK-ERK kinase pathways. DPI and Cl-amidine significantly abrogated levamisole-induced NETosis, highlighting that levamisole stimulates NETs via generation of reactive oxygen species and activation of PAD enzymes. Both levamisole and acetylcholine induced NETs through MR engagement on the surface of neutrophils and not through stimulation of TLRs. Atropine, a non-selective MR antagonist, blocked levamisole mediated NETosis. Screening experiments with pharmacologic inhibitors and neutrophils from MR knockout mice demonstrated that levamisole-induced NETosis was mediated through engagement of M3 muscarinic receptor. In a cohort of 21 patients actively using cocaine, 100% had quantifiable levamisole in urine, and sera from these patients demonstrated novel autoantibodies against NET components. In longitudinal observations, ex-vivo spontaneous NET formation correlated with periods of active cocaine use.
Conclusion: Levamisole induces NETosis through engagement of M3 muscarinic receptors on the surface of neutrophils. These findings implicate a novel interaction between the cholinergic nervous system and innate immunity in the pathogenesis of levamisole-induced autoimmunity.
To cite this abstract in AMA style:Grayson PC, Carmona-Rivera C, Waldman M, Phillips K, Subramaniam V, Thompson P, Graf JD, Kaplan MJ. Levamisole Triggers Neutrophil Extracellular Trap Formation through Muscarinic Receptors in Patients with Drug-Induced Vasculitis [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/levamisole-triggers-neutrophil-extracellular-trap-formation-through-muscarinic-receptors-in-patients-with-drug-induced-vasculitis/. Accessed May 16, 2022.
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