Increased levels of neutrophil extracellular trap remnants in the serum of patients with rheumatoid arthritis.

Neutrophil extracellular traps (NETs) comprise a unique form of non-apoptotic cell death exhibited by neutrophils, which occurs in a stepwise process termed NETosis. It has been postulated that NETosis plays an important role in the pathogenesis of autoimmune disorders. The aim of this study was to evaluate serum levels of NET remnants in patients with rheumatoid arthritis (RA), as well as potential associations between NET remnants and indicators of RA. Serum levels of myeloperoxidase (MPO)-DNA complexes (NET remnants) were examined in 74 RA patients and 50 healthy controls using a modified enzyme-linked immunosorbent assay. Associations between the levels of these complexes and indicators of RA were then statistically evaluated. RA patients exhibited significantly higher levels of MPO-DNA complexes than the healthy controls, and these levels were associated with increased neutrophil counts and positivity for rheumatoid factor (RF) and anti-citrullinated protein/peptide antibodies (ACPA). Among the 63 ACPA-positive RA patients examined, those with ACPA titers > 1600 U/mL showed significantly increased MPO-DNA levels. Receiver operating characteristic analysis determined that the area under the curve for all 74 RA patients was 0.798, with a sensitivity of 91.9% and a specificity of 56.0%, while that for the ACPA-negative patients was 0.891, with a sensitivity and specificity of 81.8% and 84.0%, respectively. The results of this study suggest that the disease status of RA is associated with increased NETosis. In particular, evaluation of serum MPO-DNA levels may comprise a useful complementary tool for discriminating RA patients from healthy individuals.

Wang W ，Peng W ，Ning X 《-》

Enhanced neutrophil extracellular trap generation in rheumatoid arthritis: analysis of underlying signal transduction pathways and potential diagnostic utility.

Sur Chowdhury C ，Giaglis S ，Walker UA ，Buser A ，Hahn S ，Hasler P ... -  《-》

Anti-citrullinated protein antibodies are associated with neutrophil extracellular trap formation in rheumatoid arthritis.

We evaluated the associations of anti-citrullinated protein antibodies (ACPAs) and serological and cytological levels of neutrophil extracellular trap (NET) formation in rheumatoid arthritis (RA) patients. Serum levels of myeloperoxidase-DNA and elastase-DNA complexes (NET remnants) were examined in 51 patients with RA and 40 healthy controls using a modified enzyme-linked immunosorbent assay. Neutrophils were isolated by density gradient centrifugation. IgG antibodies were purified by affinity chromatography. NET formation in RA and control neutrophils was assessed by microscopy in vitro. NETs were purified and co-incubated with fibroblast-like synoviocyte (FLS) cells. Interleukin (IL)-6 and IL-8 mRNA expression and protein levels in FLS cells were determined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. In RA patients, NET remnants in the peripheral circulation were higher in extremely high ACPA titers when compared to in moderate ACPA titers. And IgG antibodies containing ACPA can stimulate neutrophils to form NETs in a concentration-dependent manner. Furthermore, significantly higher expression of the pro-inflammatory cytokines IL-6 and IL-8 is detected after FLS cells interacted with NETs which derived from neutrophils stimulated with ACPA-containing IgG antibodies. Anti-citrullinated protein antibodies may enhance NET formation and contribute to inflammation development in RA by stimulating NET formation, such as by subsequent activation of FLS cells by NETs.

Wu S ，Peng W ，Liang X ，Wang W ... -  《-》

Autoantibodies to neutrophil extracellular traps represent a potential serological biomarker in rheumatoid arthritis.

Neutrophil extracellular traps (NETs) are networks of extracellular chromatin decorated with antimicrobial proteins, formed by neutrophils to entrap pathogens. NETs have been implicated in the generation of autoimmune reactions. Here, we investigate the reactivity of rheumatoid arthritis (RA) serum antibodies with NETs and explore whether anti-NET antibodies (ANETA) have a potential as biomarker in RA. To quantify ANETA, we developed an ELISA with NETs isolated from stimulated human neutrophils and verified the results by immunofluorescence staining of NETs. ANETA were detected in 22%-69% of RA sera. No significant differences were observed in the reactivity of RA sera with NETs originating from RA patients and healthy control neutrophils, nor with NETs induced by phorbol 12-myristate 13-acetate or the calcium ionophore A23187. ANETA were detected already at baseline in newly diagnosed RA patients and both increased and decreased levels were observed in samples with a median follow-up of 7 years. By ANETA ELISA, we showed that ANETA are also present in sera of patients with systemic lupus erythematosus (36%), Sjögren's syndrome (76%) and scleroderma (61%). In addition to antibodies to NETs, also the presence of NETs or NET fragments in RA sera was determined using a sandwich ELISA. Elevated levels of NETs or NET fragments were detected in 32% of the sera. To assess the potency of ANETA as a biomarker in RA, we compared ANETA positivity with other clinical features. The presence of ANETA was significantly higher in rheumatoid factor (RF)-positive patients, but did not correlate with anti-citrullinated protein antibodies (ACPA), nor with the presence of NET fragments in serum. In addition, no correlation was observed with age, gender, onset of the disease, disease activity and inflammatory markers. These findings suggest that ANETA may be an independent biomarker in RA and possibly also in other autoimmune diseases.

de Bont CM ，Stokman MEM ，Faas P ，Thurlings RM ，Boelens WC ，Wright HL ，Pruijn GJM ... -  《-》

Differential ACPA Binding to Nuclear Antigens Reveals a PAD-Independent Pathway and a Distinct Subset of Acetylation Cross-Reactive Autoantibodies in Rheumatoid Arthritis.

Lloyd KA ，Wigerblad G ，Sahlström P ，Garimella MG ，Chemin K ，Steen J ，Titcombe PJ ，Marklein B ，Zhou D ，Stålesen R ，Ossipova E ，Lundqvist C ，Ekwall O ，Rönnelid J ，Mueller DL ，Karlsson MCI ，Kaplan MJ ，Skriner K ，Klareskog L ，Wermeling F ，Malmström V ，Grönwall C ... -  《Frontiers in Immunology》