中性粒细胞胞外诱捕网放大脂多糖诱导的肺泡巨噬细胞炎症反应
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摘要
目的观察中性粒细胞胞外诱捕网(NETs)对脂多糖(LPS)诱导的肺泡巨噬细胞炎症反应的放大作用。方法采用小鼠外周血中性粒细胞分离试剂盒分离提取C57BL/6小鼠外周血中性粒细胞。采用佛波酯(PMA)(100nmol/L)诱导中性粒细胞形成NETs,采用荧光显微镜及扫描电子显微镜观察NETs。体外培养大鼠肺泡巨噬细胞NR8383,分为⑴对照组:加入等体积PBS;⑵NETs组:加入制备好的(1.7×10~6个/ml)NETs;⑶LPS组:加入1mg/L LPS;⑷LPS+NETs组:LPS联合NETs共刺激,各组均刺激12h。采用酶联免疫吸附试验(ELISA)检测细胞上清液中白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)含量。结果荧光显微镜下观察示对照组中性粒细胞核形态完整,无DNA与NETs的胞外共定位网状结构,PMA刺激组可见DNA网状结构,且与NETs共定位;扫描电子显微镜下也观察到中性粒细胞染色质呈网状结构释放至胞外,提示PMA诱导NETs形成成功。采用LPS、NETs分别刺激肺泡巨噬细胞12h,细胞上清液中的IL-1β、TNF-α含量均较对照组显著升高,LPS+NETs组上清液中的IL-1β含量较LPS组显著升高(P<0.05),上清液中的TNF-α较LPS组相比差异无统计学意义(P>0.05)。结论 NETs增加LPS诱导的肺泡巨噬细胞炎症因子释放,提示NETs可以放大LPS诱导的肺泡巨噬细胞炎症反应。
Objective To observe the amplification effect of neutrophil extracellular traps(NETs) on inflammatory response ofalveolar macrophages induced by lipopolysaccharide(LPS). Methods Neutrophils were isolated and extracted from peripheral bloodof C57 BL/6 mice by using a mouse peripheral blood neutrophil separation kit. NETs were induced by PMA(100 nmol/L). Observa-tion of NETs by fluorescence microscopy and scanning electron microscopy. NR8383 rat alveolar macrophages were cultured invitro and divided into ⑴control group:adding equal volume PBS;⑵NETs group:adding prepared(1.7×106 cell/ml) NETs;⑶LPSgroup: adding 1 mg/L LPS; ⑷LPS+NETs group:LPS combined with NETs co-stimulation. All groups were stimulated for 12 hours.Enzyme-linked immunosorbent assay(ELISA) was used to detect the contents of interleukin-1β(IL-1β) and tumor necrosis fac-tor-α(TNF-α) in the supernatant of cells. Results Fluorescence microscopy showed that the nucleus of neutrophils in the controlgroup was complete,and there was no extracellular co-localized reticular structure between DNA and NETs. DNA reticular struc-ture could be seen in PMA stimulation group and co-localized with NETs. Scanning electron microscopy also showed that thechromatin of neutrophils was released into extracellular structure,suggesting that the formation of NETs was successfully inducedby PMA. LPS and NETs were used to stimulate alveolar macrophages for 12 hours respectively,the contents of IL-1β and TNF-αin supernatant of alveolar macrophages were significantly higher than those in control group.The contents of IL-1β in supernatantof LPS+NETs group were significantly higher than those of LPS group(P<0.05). There was no significant difference in TNF-α insupernatant between LPS +NETs group and LPS group(P >0.05). Conclusion NETs can amplify LPS-induced inflammation inalveolar macrophages. NETs increased LPS-induced alveolar macrophage inflammatory factor release,suggesting that NETs canamplify LPS-induced alveolar macrophage inflammatory response.
Objective To observe the amplification effect of neutrophil extracellular traps(NETs) on inflammatory response ofalveolar macrophages induced by lipopolysaccharide(LPS). Methods Neutrophils were isolated and extracted from peripheral bloodof C57 BL/6 mice by using a mouse peripheral blood neutrophil separation kit. NETs were induced by PMA(100 nmol/L). Observa-tion of NETs by fluorescence microscopy and scanning electron microscopy. NR8383 rat alveolar macrophages were cultured invitro and divided into ⑴control group:adding equal volume PBS;⑵NETs group:adding prepared(1.7×106 cell/ml) NETs;⑶LPSgroup: adding 1 mg/L LPS; ⑷LPS+NETs group:LPS combined with NETs co-stimulation. All groups were stimulated for 12 hours.Enzyme-linked immunosorbent assay(ELISA) was used to detect the contents of interleukin-1β(IL-1β) and tumor necrosis fac-tor-α(TNF-α) in the supernatant of cells. Results Fluorescence microscopy showed that the nucleus of neutrophils in the controlgroup was complete,and there was no extracellular co-localized reticular structure between DNA and NETs. DNA reticular struc-ture could be seen in PMA stimulation group and co-localized with NETs. Scanning electron microscopy also showed that thechromatin of neutrophils was released into extracellular structure,suggesting that the formation of NETs was successfully inducedby PMA. LPS and NETs were used to stimulate alveolar macrophages for 12 hours respectively,the contents of IL-1β and TNF-αin supernatant of alveolar macrophages were significantly higher than those in control group.The contents of IL-1β in supernatantof LPS+NETs group were significantly higher than those of LPS group(P<0.05). There was no significant difference in TNF-α insupernatant between LPS +NETs group and LPS group(P >0.05). Conclusion NETs can amplify LPS-induced inflammation inalveolar macrophages. NETs increased LPS-induced alveolar macrophage inflammatory factor release,suggesting that NETs canamplify LPS-induced alveolar macrophage inflammatory response.
引文
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[13]Jiao Y,Li Z,Loughran PA,et al. Frontline Science:Macrophage-derived exosomes promote neutrophil necroptosis followinghemorrhagic shock[J]. Journal of Leukocyte Biology,2018,103(2):175-183.
[14]方铭,邓君,黄亮. 62例创伤性休克治疗体会[J].江西医药,2012,47(6):500-501.
[15]Warnatsch A,Ioannou M,Wang Q,et al. Inflammation. Neutrophilextracellular traps license macrophages for cytokine production inatherosclerosis[J]. Science,2015,349(6245):316-320.
[16]Segal AW. How neutrophils kill microbes[J]. Annual Review ofImmunology,2005,23(:197-223.
[17]Nathan C. Neutrophils and immunity:challenges and opportunities[J]. Nature reviews. Immunology,2006,6(3):173-182.
[18]Keshari RS,Verma A,Barthwal MK,et al. Reactive oxygenspecies-induced activation of ERK and p38 MAPK mediatesPMA-induced NETs release from human neutrophils[J]. Journal ofCellular Biochemistry,2013,114(3):532-540.
[19]谢建平,白建文.中性粒细胞胞外捕获在急性肺损伤发病机制中的研究进展[J].中国急救医学,2017,37(9):860-863.
[20]Chen L,Zhao Y,Lai D,et al. Neutrophil extracellular trapspromote macrophage pyroptosis in sepsis[J]. Cell Death&Disease,2018,9(6):597.
[2]Brinkmann V,Zychlinsky A. Beneficial suicide:why neutrophils dieto make NETs[J]. Nature reviews. Microbiology,2007,5(8):577-582.
[3]Kaplan MJ,Radic M. Neutrophil extracellular traps:double-edgedswords of innate immunity[J]. Journal of Immunology,2012,189(6):2689-2695.
[4]Papayannopoulos V,Zychlinsky A. NETs:a new strategy for usingold weapons[J]. Trends in immunology,2009,30(11):513-521.
[5]Cheng OZ,Palaniyar N. NET balancing:a problem in inflammatorylung diseases[J]. Front Immunol,2013,4:1.
[6]Grégoire M,Uhel F,Lesouhaitier M,et al. Impaired efferocytosisand neutrophil extracellular trap clearance by macrophages inARDS[J]. The Eur Respir J,2018,52(2).
[7]邵强,李勇,刘芬,等.脂多糖对肺泡巨噬细胞自噬水平的影响[J].江西医药,2012,47(2):118-120.
[8]Wynn TA,Vannella KM. Macrophages in Tissue Repair,Regeneration,and Fibrosis. Immunity,2016,44(3):450-462.
[9]陈琳,徐国海,陈勇.右美托咪定对脓毒症大鼠肺组织HMG B1表达的影响[J].江西医药,2016,51(5):415-417,423.
[10]Fan J,Li Y,Vodovotz Y,et al. Neutrophil NAD(P)H oxidase isrequired for hemorrhagic shock-enhanced TLR2 up-regulation inalveolar macrophages in response to LPS[J]. Shock,2007,28(2):213-218.
[11]Fan J,Malik AB. Toll-like receptor-4(TLR4)signaling augmentschemokine-induced neutrophil migration by modulating cellsurface expression of chemokine receptors[J]. Nature medicine,2003,9(3):315-321.
[12]Fan J,Li Y,Vodovotz Y,et al. Hemorrhagic shock-activatedneutrophils augment TLR4 signaling-induced TLR2 upregulationin alveolar macrophages:role in hemorrhage-primed lunginflammation[J]. American journal of physiology. Lung cellular andmolecular physiology,2006,290(4):L738-738L746.
[13]Jiao Y,Li Z,Loughran PA,et al. Frontline Science:Macrophage-derived exosomes promote neutrophil necroptosis followinghemorrhagic shock[J]. Journal of Leukocyte Biology,2018,103(2):175-183.
[14]方铭,邓君,黄亮. 62例创伤性休克治疗体会[J].江西医药,2012,47(6):500-501.
[15]Warnatsch A,Ioannou M,Wang Q,et al. Inflammation. Neutrophilextracellular traps license macrophages for cytokine production inatherosclerosis[J]. Science,2015,349(6245):316-320.
[16]Segal AW. How neutrophils kill microbes[J]. Annual Review ofImmunology,2005,23(:197-223.
[17]Nathan C. Neutrophils and immunity:challenges and opportunities[J]. Nature reviews. Immunology,2006,6(3):173-182.
[18]Keshari RS,Verma A,Barthwal MK,et al. Reactive oxygenspecies-induced activation of ERK and p38 MAPK mediatesPMA-induced NETs release from human neutrophils[J]. Journal ofCellular Biochemistry,2013,114(3):532-540.
[19]谢建平,白建文.中性粒细胞胞外捕获在急性肺损伤发病机制中的研究进展[J].中国急救医学,2017,37(9):860-863.
[20]Chen L,Zhao Y,Lai D,et al. Neutrophil extracellular trapspromote macrophage pyroptosis in sepsis[J]. Cell Death&Disease,2018,9(6):597.