Effect Of Neutrophil Extracellular Traps On Pathologic Process In Systemic Lupus Erythematosus

Background:Systemic lupus erythematosus(SLE) is a systemic autoimmune disease involved multiple systems and multi-organs, charactered as antinuclear antibodies producing and immune complex depositing in small vascular. The basic pathological changes of SLE are connective tissue inflammation, immune complex deposition and vascular lesions.Neutrophils play a critical role in defense against bacteria. Neutrophils are the most abundant cells in peripheral blood leukocytes and have the short lifespan. In physiological conditions, apoptosis is the main way of neutrophil death. A new way of death named NETosis was discovered in 2004, which is different from apoptosis and necrosis. NETs(neutrophil extracellular traps), a Net-like structure made up of DNA, histones, antibacterial peptides and elastase etc, is also a new sterilization method of neutrophils. NETs was produced when NETosis occurred.Studies have shown that some nuclear materials and antimicrobial proteins exposed on the cell surface during NETs producing, which may become the auto-antigens, inducing the production of antibodies, then forming immune complex, and stimulating the secretion of certain inflammatory cytokines. So NETs may participate in the pathogenesis of systemic lupus erythematosus.Objectives:To analyse the level of NETs in SLE group and the effect of SLE plasma on generation of NETs. To make clear the relationship between NETs and some clinical indexes. And to clarify the effect of NETs on production of some inflammatory in pathologic process of SLE.Methods:1. A total of 44 patients with SLE were investigated as SLE group and peripheral venous blood was collected and treated with heparin anticoagulation. Polymorphonuclear leucocyte(PMN) were sterilly separated, and plasma was conserved at-80℃. Clinical materials of the patients were collected. 42 healthy volunteers matched in age and sex were as control subjects.2. NETs detection: the level of NETs was quantitatively detected by fluorospectrophotometry, and statistic analyse the difference of level of NETs between SLE group and healthy control group. Then assay the correlation between the level of NETs and ESR, and CRP, and anti-ds-DNA, and anti-histone, and anti-nucleosome, and SLEDAI, respectively.The method of indirect immunofluorescence was used to detect nuclear DNA and neutrophil elastase(NE), the components of NETs, and the cells were observed by laser scanning confocal microscope.3. Concentration of neutrophil elastase in plasma was detected by enzyme-linked immunosorbent assay. The statistical difference of NE level was assayed between SLE group and healthy control group. Then assay the correlation between the level of NE and ESR, and CRP, and anti-ds-DNA, and anti-histone, and anti-nucleosome, and SLEDAI, respectively.4. Human peripheral leucocytes from 33 healthy donors were cultured with SLE mixed plasma from 30 patients and healthy mixed plasma from 30 donors respectively for 24 hours in 5% CO2 at 37°C. Then the level of NETs and the concentration of IL-17, IL-6 and IFN-αwere detected and the differences were analysed between two groups.5. PMN was stimulated by PMA with the different concentration of 0, 12.5, 25 or 50 n M to generate different levels of NETs(the level of NETs increased with the elevation of PMA concentration)]. Then the PMN with NETs was used to stimulate PBMCs for 24 h and 48 h and the concentrations of IL-17, IL-6 and IFN-αin supernate were detected. Analyse the effects on the secretion of IL-17, IL-6 and IFN-αby PBMCs with different level of NETs and stimulating time.Result:1.The fluorescence intensity of NETs in SLE group and control group were [(240.82±139.20)vs(172.57±134.61), P=0.002)]. The level of NETs was significantly increased in SLE group.2.The level of NETs in SLE patients was positively correlated with SLEDAI(R=0.402, P=0.020), but had no obvious correlation with anti-ds DNA( R=-0.065, P=0.675), anti-histone(R=0.034, P=0.827), anti-nucleosome(R=-0.268,P=0.078), CRP(R=0.122, P=0.504) and ESR(R=0.291, P= 0.101), respectively.3.The concentration of NE in plasma of SLE group and control group was [(102.02±47.02)vs(61.76±22.25), P = 0.000)]. The concentration of NE in the SLE group was significantly higher than that in control group.4.The concentration of NE in SLE patients was positively correlated with SLEDAI(R=0.341, P=0.032), but had no obvious correlation with anti-ds DNA( R=0.177, P=0.499),anti-histone(R=-0.291, P=0.167), anti-nucleosome(R=0.202,P=0.343), CRP(R=-0.437,P=0.07)and ESR(R=-0.021,P=0.926), respectively.5. Perpheral leucocytes were cultured for 24 h with SLE mixed plasma or HC mixed plasma,( 1) the fluorescence intensity of NETs was [(88.06±47.01) vs(63.89±50.49), P=0.000). The level of NETs was significant different between SLE and control groups. The results showed the NETs increase of PMN treated with SLE mixed plasma.(2)Comparation of the concentrations of inflammatory cytokines between two groups:The concentrations of IL-17 was IL-17 [5.77(2.42,12.11)pg/ml vs 2.99(1.82,3.87)pg/ml,P=0.002],IL-6 [606.69(233.64,916.76) pg/ml vs 93.71( 13.09, 181.54) pg/ml, P=0.000],IFN-α[( 98.32±53.13) pg/ml vs( 10.92±10.11) pg/ml, P=0.000]. The concentration of three inflammatory cytokines were significant different between two groups. The results showed that the concentration of IL-17, IL-6 and IFN-αincreased significantly in PBMCs treated with plasma of SLE.6. After PBMCs were treated with different levels of the NETs for 24 and 48 h, the concentration of IL-6 and IFN-αin the culture supernatant was significantly higher than that of untreated group. Otherwise, the increase of IL-17 was not significant.Conclusions:1. NETs in female patients with SLE were significantly higher than that in the control group. It indicates that NETs formation increase in patients with SLE, and that NETs may participate in the pathological process of SLE.2. The level of NETs in SLE patients had positive correlation with SLE disease activity index suggest that NETs may be an evaluation index of SLE severity. But our results show that NETs have no correlation with some autoantibodies.3. Some factors in plasma environment of SLE can promote NETs formation of PMNs from healthy controls and inflammatory cytokines as IL-17,IL-6,IFN-αmay be an effective fator.4. NETs produced from PMN treated with PMA can stimulate IL- 6 and IFN-αsecretion. It suggests that NETs may be a cause of SLE inflammatory lesions.