Neutrophil-mediated mechanisms of damage and in-vitro protective effect of colchicine in non-vascular Behçet's syndrome

Abstract

Behçet's syndrome (BS) is a systemic vasculitis with several clinical manifestations. Neutrophil hyperactivation mediates vascular BS pathogenesis, via both a massive reactive oxygen species (ROS) production and neutrophil extracellular traps (NETs) release. Here, we investigated neutrophil-mediated mechanisms of damage in non-vascular BS manifestations and explored the in-vitro effects of colchicine in counteracting these mechanisms. NETs and intracellular ROS production was assessed in blood samples from 80 BS patients (46 with active non-vascular BS, 34 with inactive disease) and 80 healthy controls. Moreover, isolated neutrophils were incubated for 1 h with an oxidating agent [2,2'-azobis (2-amidinopropane) dihydrochloride; 250 nM] and the ability of pure colchicine pretreatment (100 ng/ml) to counteract oxidation-induced damage was assessed. Patients with active non-vascular BS showed remarkably increased NET levels [21.2, interquartile range (IQR) = 18.3-25.9 mU/ml] compared to patients with inactive disease (16.8, IQR = 13.3-20.2 mU/ml) and to controls (7.1, IQR = 5.1-8.7 mU/ml, p < 0.001]. Also, intracellular ROS tended to increase in active BS, although not significantly. In active non-vascular BS, NETs correlated with neutrophil ROS production (p < 0.001) and were particularly increased in patients with active mucosal (p < 0.001), articular (p = 0.004) and gastrointestinal symptoms (p = 0.006). In isolated neutrophils, colchicine significantly reduced oxidation-induced NET production and cell apoptosis, although not via an anti-oxidant activity. Neutrophil-mediated mechanisms might be directly involved in non-vascular BS, and NETs, more than ROS, might drive the pathogenesis of mucosal, articular and intestinal manifestations. Colchicine might be effective in counteracting neutrophils-mediated damage in BS, although further studies are needed.

Keywords: autoimmunity; human; neutrophils; reactive oxygen species; vasculitis.

FIGURE 1

Neutrophil extracellular trap (NET) levels (a) and neutrophilic reactive oxygen species (ROS) production (b) in BS patients with active non‐vascular disease manifestations with inactive disease and in healthy controls (HC). (c) Representative confocal microscope images of neutrophils prone to extrude DNA in blood samples from HC, from BS patients active non‐vascular BS manifestations and with inactive disease, stained with propidium iodide. NETs can be observed as the DNA that is going to be extruded from the cell in figure (white arrows). In the microscope images of samples from BS patients with active disease (c, central image), a diffuse opacity, organic waste and cellular debris can be detected on the slide, due to the massive neutrophils cell death. Magnification ×630

FIGURE 2

Correlation between neutrophil extracellular trap (NET) levels and intracellular neutrophils reactive oxygen species (ROS) production in BS patients with active non‐vascular disease manifestations with inactive disease and in healthy controls

FIGURE 3

Neutrophil extracellular trap (NET) levels (a–c) and neutrophils ROS production (d–f), stratified according to active BS disease manifestations

FIGURE 4

Representative confocal microscope images (a) of neutrophil extracellular traps (NETs) in neutrophils untreated, incubated with 2,2′‐azobis(2‐amidinopropane) dihydrochloride (AAPH) (250 mM) for 1 h and pretreated with colchicine (100 ng/ml) for 1 h before AAPH treatment. NET levels (b), cell viability (c) and mitochondrial polarization (d) in neutrophils untreated, incubated with AAPH or pretreated with colchicine before AAPH incubation