| Background:Systemic lupus erythematosus(SLE)is a common autoimmune disease.It is characterized by a large number of antinuclear antibodies and immune complexes.Its pathogenesis is not clear,and there is a lack of radical cure in clinic.The research on its pathogenesis is particularly important.Recently,it is widely believed that the dysfunction of apoptotic cells clearance in vivo is a potential source of endogenous nuclear antigen.Neutrophils as the main cells of the innate immune system,under physiological conditions mainly through apoptosis in death,studies found that neutrophils generation of neutrophil extracellular traps(NETs)is another potential source of nuclear antigen.Studies have shown that NETs levels in SLE patients are significantly elevated and correlated with disease activity.NETs is associated with increased production and clearance disorders,but the specific mechanism is not yet clear.Integrin is a member of cell adhesion molecules.It regulates cell migration,proliferation and apoptosis by participating in "bidirectional signal transduction" between extracellular matrix and extracellular matrix,and is involved in the pathological and physiological process of organism.The macrophage 1 antigen(Mac-1)is highly expressed in neutrophils,and the gene ITGAM that encodes Mac-1 alpha chain belongs to the susceptible gene of lupus.The antibacterial peptide LL-37 is both a NETs component and a ligand of Mac-1,which can induce the activation of Mac-1 on neutrophils.So,does Mac-1 participate in the formation or removal of NETs? What is the relationship between Mac-1 and NETs can help to discover the cause of the increase in the level of NETs in SLE,which may reveal the formation mechanism of SLE autoantibodies.Objectives:To assay the expression of Mac-1 on neutrophils,monocytes and T cells in SLE patients.To analyze the correlation between Mac-1 and NETs,further,and the role of Mac-1 in the formation of NETs,clarify whether mac-1 is involved in NETs mediated pathology in SLE.To elucidate whether mac-1 is involved in NETs mediated pathogenesis in SLE.Methods:1.Collect 49 patients with SLE and 37 healthy volunteers fasting venous blood,heparin,separate neutrophils by single density gradient centrifugation for the detection of NETs,plasma stored at-80℃.EDTA-K2 anticoagulant blood to detect the expression of Mac-1.The clinical data of SLE patients were collected at the same time2.NETs detection: the level of NETs was detected by fluorospectrophotometry and enzyme-linked immunosorbent assay,and analyse the difference of level of NETs between SLE group and healthy control group、Antinuclear antibody positive group and negative group.Then analyses the correlation between the level of NETs and ESR,CRP,anti-ds-DNA,C3,C4,SLEDAI,respectively.3.Concentration of IL-6,TNF-α and INF-α inflammatory cytokines in plasma was detected by enzyme-linked immunosorbent assay.The statistical difference of IL-6,TNF-α and INF-α level was assayed between SLE group and healthy control group.Then analyses the correlation between the level of IL-6、TNF-α、INF-α and NE,MPO,respectively.4.The expression of integrin Mac-1 on peripheral leukocytes was detected by flow cytometry.Analyse the difference of expression of Mac-1 between SLE group and healthy control group.Then analyses the correlation between the level of Mac-1 and NE,MPO,ESR,CRP,anti-ds-DNA,C3,C4,SLEDAI,respectively.5.SLE and healthy plasma were prepared into respectively mixed plasma,with different concentrations of Mac-1 ligand sICAM-1 were treated from SLE patients and healthy subjects,neutrophils 24 h,48h,using fluorescence spectrophotometry quantitative detection of NETs levels.Then the effect of analysis of sICAM-1 on the formation of NETs levels.Result:1.The concentrations of NE and MPO in plasma of SLE group(45)and control group(24)was [NE(72.7±33.76 vs 38.94±6.89,P= 0.000;MPO 2.51(1.29,5.43)vs 0.99(0.86,1.64),P= 0.000].The levels of NE and MPO in SLE group were significantly higher than in control group.The fluorescence intensity of NETs and the concentration of NE in plasma(R=0.881,P=0.000)had significant positive correlation.However,the fluorescence intensity of NETs and the concentration of MPO in the plasma(R=0.49,P=0.075)had a positive correlation trend,but no significant difference.2.Concentrations of NE and MPO in plasma from patients with SLE of anti-dsDNA antibody positive group(20)and negative group(8)were [NE(88.3±21.25)vs(44.97±21.76),P=0.000;MPO 4.92(2.26,44.32)vs 1.93(1,7.17),P=0.042].The levels of NE and MPO in anti-dsDNA antibody positive group were significantly higher than in negative group.3.Concentrations of NE and MPO in plasma from patients with SLE of anti-histone antibody positive group(13)and negative group(15)were [NE(86.85±27.3 vs 66.46±27.45,P=0.062;MPO 8.22(2.66,48.68)vs 3.71(1.95,5.37),P=0.020].Compared with the negative group,the level of NE in the anti-histone antibody positive group have a increased trend.Anti-histone antibody-positive MPO levels were significantly higher than the negative group.4.Concentrations of NE and MPO in plasma of patients with SLE of anti-nucleosome antibody positive group(15)and negative group(13)were[NE(80.5±26.59)vs(70.65±31.68),P=0.379;MPO 18.21(3.7,48.67)vs 2.66(1.22,4.51),P=0.015].There was no significant difference in NE levels between the two groups.However,the level of MPO in anti-nucleosome antibody positive group was significantly higher than that in negative group.5.The concentration of NE in SLE patients was positively correlated with SLEDAI(R= 0.579,P= 0.001),but negatively correlated with the levels of complement C3 and C4(R=-0.584,P=0.001;R=-0.475,P=0.011);but had no obvious correlation with urinary protein level(R=0.058,P=0.771),CRP(R=0.314,P=0.104)and ESR(R=0.203,P=0.248),respectively.The level of MPO in plasma of SLE patients was positively correlated with urinary protein level(R=0.579,P=0.001),but had no obvious correlation with ESR(R= 0.369,P=0.053)、 C3(R=-0.249,P=0.201)、 C4(R=-0.152,P= 0.439)、CRP(R=0.272,P=0.162)and SLEDAI score(R=0.163,P=0.39),respectively.6.The levels of IL-6,TNF-α and INF-α in the plasma of the SLE group(30)and control group(22)were[IL-6(14.69±5.2)vs(9.47±2.3),P=0.004;TNF-α(33.58±10.53)vs(23.04±5.36),P= 0.01;IFN-α [33.86(14.7,153.47)vs 12.38(9.67,24.63),P=0.000].The concentration of IL-6,TNF-α and INF-α in the SLE group was significantly higher than in control group.7.The levels of NE in SLE patients had no obvious correlation with the concentration of inflammatory cytokines IL-6(R=0.316,P=0.095)、INF-α(R=0.191,P=0.321)、TNF-α(R=0.291,P=0.126),respectively.The levels of MPO in SLE patients had no obvious correlation with the concentration of inflammatory cytokines IL-6(R=0.263,P=0.168)、IFN-α(R=0.059,P=0.762)、TNF-α(R=0.182,P=0.345),respectively.8.The positive expression rate of Mac-1 on PMN of SLE group(49)and controls group(37)was [(99.79±0.31)vs(99.93±0.28),P=0.037].No clinical significance.The average expression of Mac-1 in single cells was[(28350.00±11283.10)vs(16818.15±5073.23),P=0.000].The expression of Mac-1 on PMN of SLE group was significantly higher than in control group.9.The positive expression rate of Mac-1 on monocytes of SLE group(49)and controls group(37)was [(97.89±1.89)vs(96.87±2.09),P=0.024].No clinical significance.The average expression of Mac-1 in single cells was [(26701.77±12191.20)vs(15074.16±5231.68),P=0.000].The expression of Mac-1 on monocytes of SLE group was significantly higher than in control group.10.The positive expression rate of Mac-1 on T lymphocytes of SLE group(49)and controls group(37)was [(41.23±13.23)vs(28.84±6.97),P=0.000].The average expression of Mac-1 in single cells was [(592.68±253.41)vs(356.64±134.12),P=0.000].The expression of Mac-1 on T lymphocytes of SLE group was significantly higher than in control group.11.The expression of Mac-1 on PMN of SLE had no obvious correlation with the SLEDAI score(R=-0.075,P=0.704),C3(R=-0.153,P=0.437)、C4(R=-0.108,P=0.568)、CRP(R=0.301,P=0.12),ESR(R= 0.059,P=0.766),urinary protein level(R=-0.207,P=0.291),NE(R=0.022,P=0.912)and MPO(R=0.195,P=0.319),respectively.12.Different concentrations of sICAM-1(0、250、500、750ng / ml)were used to treat PMN from normal SLE/ control for 24/48 h,the level of NETs did not change with the increase of sICAM-1 concentration at different time points.Conclusions:1.The NE level in SLE plasma was positively correlated with disease activity,negatively correlated with complement,and plasma MPO concentration positively correlated with urine protein level.The levels of NE and MPO in anti-dsDNA antibody positive group were significantly higher than in negative group.MPO levels in anti-histone antibody positive group and anti-nucleosome antibody positive group were significantly higher than in corresponding negative group.This indicates that NETs may promote the production of autoantibodies in SLE,and MPO can be used as an index to assess the severity of SLE nephropathy.2.The levels of inflammatory cytokines IL-6,TNF-α and INF-α in patients with SLE were significantly higher than in healthy controls,but not correlated with NETs levels,suggesting that the generation of NETs may not be related to the above-mentioned production of inflammatory cytokines in SLE patients.3.The expression of Mac-1 in peripheral blood mononuclear cells,PMN cells and T cells in SLE group were significantly higher than in healthy control group.It is speculated that Mac-1 may be involved in the pathogenesis of SLE,but may not be related to the formation of NETs.4.sICAM-1 activation of Mac-1 may not mediate the production of NETs... |
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