Abstract
BackgroundStudies have suggested that soluble factors in plasma from patients with active (aBD) and inactive (iBD) Behçet’s disease (BD) stimulate neutrophil function. Soluble CD40 ligand (sCD40L) is an important mediator of inflammation in BD. Its expression and effect on neutrophil oxidative burst and neutrophil extracellular trap (NET) release have not been characterized. In this study, we sought to investigate the role of plasma and the CD40L pathway on NET release and the oxidative burst profile in patients with aBD and iBD.MethodsNeutrophils and peripheral blood mononuclear cells (PBMCs) were obtained from patients with aBD (n = 30), patients with iBD (n = 31), and healthy control subjects (HCs; n = 30). sCD40L plasma concentration was determined in individual samples. A pool of plasma for each group was created. In some experiments, plasma pools were treated with recombinant CD40 (rhCD40-muIg) for sCD40L blockade. NET release and H2O2/O2− production were determined after stimulation with phorbol 12-myristate 13-acetate, sCD40L, or plasma pool. Flow cytometric analysis was performed to evaluate the expression of (1) CD40, Mac-1, and phosphorylated NF-κB p65 on neutrophils and monocytes and (2) CD40L on activated T cells and platelets. CD40L gene expression in PBMCs was determined by qRT-PCR.ResultssCD40L plasma levels were significantly higher in patients with iBD (median 17,234, range 2346–19,279 pg/ml) and patients with aBD (median 18,289, range 413–19,883 pg/ml) than in HCs (median 47.5, range 33.7–26.7 pg/ml; p < 0.001). NET release was constitutively increased in BD compared with HC. NET release and H2O2/O2− were higher after stimulation with sCD40L or BD plasma and decreased after sCD40L blockade. Mac-1 expression was constitutively increased in neutrophils of patients with aBD (88.7 ± 13.2% of cells) and patients with iBD (89.2 ± 20.1% of cells) compared with HC (27.1 ± 18.8% of cells; p < 0.01). CD40 expression on phagocytes and CD40L expression on platelets were similar in the three groups. PBMCs as well as nonactivated and activated CD4+ T cells from patients with BD showed higher CD40L expression.ConclusionsPlasma from patients with aBD exerts a stimulus on NET release and oxidative burst, probably induced by sCD40L.
Highlights
Studies have suggested that soluble factors in plasma from patients with active and inactive Behçet’s disease (BD) stimulate neutrophil function
We investigated the role of plasma from patients with BD and Soluble CD40 ligand (sCD40L) on neutrophil extracellular trap (NET) release and oxidative burst profiles in neutrophils and peripheral blood mononuclear cells (PBMCs) from patients with active Behçet’s disease and patients with inactive Behçet’s disease
To determine whether the increased production of reactive oxygen species by BD neutrophils could be a result of increased expression of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits, we evaluated the level of subunit expression by both messenger RNA and protein analysis
Summary
Studies have suggested that soluble factors in plasma from patients with active (aBD) and inactive (iBD) Behçet’s disease (BD) stimulate neutrophil function. Several pieces of evidence point to participation of neutrophils in BD, evaluation of neutrophil extracellular trap (NET) formation in BD has not been investigated. It is unclear whether phagocyte activation in patients with BD occurs constitutively, mainly in those carrying HLA-B51 [3], or if it is secondary to an as yet unknown stimulus [10, 11]. Has not been defined whether the neutrophil hyperactive state is induced by some soluble serum or tissue factor or if BD neutrophils or other cell subtypes produce soluble inflammatory mediators carried by plasma, as previously suggested for specific phagocyte functions, such as chemotaxis [10] and oxidative burst [11]
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