Serum amyloid A induces the formation of neutrophil extracellular traps via Toll like receptor 4 in rheumatoid arthritis / 中华风湿病学杂志

ABSTRACT

Objective To investigate the neutrophil extracellular traps (NETs) formation and their molecular mechanisms induced by serum amyloid A (SAA) in rheumatoid arthritis (RA).Methods Neutrophils were isolated from peripheral blood of RA and healthy volunteers.① Neutrophils were cultured in vitro,the formation of NETs was observed and their percentage was calculated.② Neutrophils were cultured in vitro,divided into six groupscontrol,SAA,[SAA+anti-Toll like receptro4 (TLR4)-Ab],LPS,(LPS+anti-TLR4-Ab) and anti-TLR4-Ab.Appropriate stimulation was conducted for each group.NETs formation and their percentages were investigated.The concentration of DNA in supernatant was detected by fluorescent staining.F test and t test were used for statistical analysis.Results ① The purification of isolated neutrophils was higher than 95％.The network which was collocated with the spreading neutrophils nucleus and neutrophil elastase under the microscope,was NETs.In the RA group,the formation of NETs induced by SAA was significantly more than control [(19.1±0.8) vs (7.4±0.5),t=12.30,P＜0.05].② However,after pretreated with anti-TLR4 antibody,NETs formation was significantly less than the SAA group [(5.7±0.4) vs (14.7±1.1),t=7.825,P＜0.05].Moreover,the fluorescence intensity of DNA in supernatant was significantly higher in SAA group than that of anti-TLR4-Ab pretreatment group [(18.7 ±0.7) vs (12.9±0.8),t=5.552,P＜0.05].The concentration of DNA in supernatant of SAA group was higher than that of anti-TLR4-Ab pretreatment group [(36.9±1.3) μg/ml and (16.3±0.6) μ,g/ml,t=14.41,P＜0.05].Conclusion SAA can induce the formation of NETs from neutrophils by binding to TLR4 in RA.