Neutrophil extracellular traps as a potential source of autoantigen in cocaine-associated autoimmunity

Abstract

Objective: Exposure to illicit cocaine and its frequent adulterant, levamisole, is associated with ANCAs targeting neutrophil elastase (NE), neutropenia and vasculitic/thrombotic skin purpura. The mechanisms of cocaine/levamisole-associated autoimmunity (CLAA) are unknown. The aim of this study was to assess the ability of cocaine and levamisole to induce the release of neutrophil extracellular traps (NETs), a potential source of autoantigen and tissue injury.

Methods: We performed quantitative and qualitative assessment of NET formation in neutrophils from healthy donors exposed to either drug in vitro . In addition, IgG from sera of individuals with CLAA (CLAA-IgG) was assessed for its ability to enhance formation of, and to bind to, drug-induced NETs.

Results: Both cocaine and levamisole could induce formation of NETs enriched in NE and, potentially, inflammatory mitochondrial DNA. Both drugs could also augment simultaneous release of B cell-activating factor belonging to the TNF family (BAFF). CLAA-IgG, but not IgG from healthy individuals, could potentiate drug-induced NETosis. Furthermore, CLAA-IgG, but not ANCA + control IgG, bound to drug-induced NETs in a pattern consistent with NE targeting.

Conclusion: Both cocaine and levamisole may contribute to the development of ANCAs by inducing release of potentially inflammatory NETs in association with NE autoantigen and BAFF. Enhancement of drug-induced NET release by CLAA-IgG provides a potential mechanism linking vasculitis/pupuric skin disease to acute drug exposure in patients with CLAA. Further study of this under-recognized form of autoimmunity will be likely to provide mechanistic insight into ANCA-associated vasculitis and other diseases associated with NETosis.

Keywords: anti-neutrophil cytoplasm antibody; autoantibodies; autoantigens; drug-induced rheumatic disease; neutrophils; vasculitis.

Fig. 1

Cocaine and levamisole induce neutrophil extracellular trap formation (A) IF microscopic images showing DNA (blue) and neutrophil elastase (NE, red) staining of neutrophils exposed to cocaine, levamisole or PMA before fixation and permeabilization. Scale = 10 μm. Insets are digitally enlarged. (B and C) DNA (B) and NE release (C) were quantified after enzymatic detachment of NETs (n = 16 experiments for B, and n = 6 for C). (D) Computer-aided analysis of NE/DNA intensity in images represented in A (median values indicated). (E) DNA release in cultures identical to those in A–D pre-incubated for 60 min with inhibitors of PAD4 (Cl-amidine), NADPH oxidase (apocynin), mitochondrial ROS (TTFA) or both NADPH oxidase and mitochondrial ROS (DPI) (n = 3–9 experiments). (F) Ratios of mitochondrial (16S) DNA/nuclear (18S) DNA in released NETs as determined by quantitative PCR (n = 10 experiments). (G) BAFF release by neutrophils stimulated for 16 h as determined by ELISA (n = 5–7 experiments). *P < 0.05, **P < 0.01 and ***P < 0.001, Student’s paired t-test vs control (unless otherwise indicated). For D, one-way analysis of variance with correction for multiple comparisons was used. Shown are mean values and s.d.s, except for D, which shows individual and median values. DPI: diphenyleneiodonium; NADPH: nicotinamide adenine dinucleotide phosphate; NET: neutrophil extracellular trap; PAD4: peptidyl arginine deiminase, type 4; PMA: phorbol myristate acetate; TTFA: thenoyltrifluoroacetone.

Fig. 2

Cocaine/levamisole-associated autoimmunity-IgG amplifies drug-induced neutrophil extracellular trap formation and binds to neutrophil extracellular trap-associated antigens DNA release from neutrophils that were pre-incubated with IgG isolated from HC, AC or CLAA subjects, then stimulated with cocaine (A), levamisole (B) or PMA (C). The results are derived from three or four independent experiments with IgG from three individuals for each condition. (D) Serum anti-NE IgG levels in HC, AC and CLAA subjects. (E) IF microscopic images showing DNA (green) and IgG (red) staining of unstimulated and levamisole-stimulated neutrophils incubated with HC, AC or CLAA sera. Scale = 10 μm. Insets are digitally enlarged. Images are representative of at least three independent experiments for each condition. *P < 0.05, **P < 0.01 and ***P < 0.001, Student’s paired t-test vs control (unless otherwise indicated). Shown are mean values and s.d.s, or mean and individual values (D). AC: ANCA control; CLAA: cocaine/levamisole-associated autoimmunity; HC: healthy control; NE: neutrophil elastase; NET: neutrophil extracellular trap; PMA: phorbol myristate acetate.