陳 清

(湖北省武漢市醫(yī)療救治中心感染科 430000)

論著·臨床研究

IL-6和IL-13與EV71型腸道病毒感染手足口病患者發(fā)病的相關(guān)性

陳 清

(湖北省武漢市醫(yī)療救治中心感染科 430000)

目的觀察腸道病毒71型(EV71)感染手足口病(HFMD)患者血清白細(xì)胞介素 (IL)-6和IL-13水平變化情況及其臨床意義，并對(duì)血清IL-6和IL-13細(xì)胞來(lái)源進(jìn)行初步的探討。方法采用酶聯(lián)免疫吸附試驗(yàn)(ELISA)法測(cè)定HFMD患者和健康對(duì)照血清中IL-6和IL-13水平，比較不同嚴(yán)重程度的HFMD患者血清中IL-6和IL-13水平，并同時(shí)檢測(cè)HFMD患者治療后血清IL-6和IL-13水平變化情況。流式細(xì)胞術(shù)(FCM)分析外周血單個(gè)核細(xì)胞(PBMC)中單核細(xì)胞分泌IL-6和T細(xì)胞分泌IL-13的情況。EV71病毒感染健康人單核細(xì)胞和T細(xì)胞后，F(xiàn)CM檢測(cè)單核細(xì)胞分泌IL-6和T細(xì)胞分泌IL-13變化情況。結(jié)果EV71感染HFMD患者血清 IL-6和IL-13水平分別為(79.63±29.45)pg/mL和(36.45±15.13)pg/mL，健康對(duì)照組血清IL-6和IL-13水平分別為(27.26±7.82)pg/mL和(13.46±3.14)pg/mL，HFMD患者血清IL-6和IL-13水平顯著高于健康對(duì)照者(P<0.01)，同時(shí)，隨著HFMD患者疾病嚴(yán)重程度增加，血清IL-6和IL-13水平亦逐漸升高(P<0.01)。治療后HFMD患者血清中IL-6和IL-13水平分別為(48.23±23.14)pg/mL和(23.25±9.63)pg/mL，與治療前相比顯著降低(P<0.01)。HFMD患者外周血單核細(xì)胞分泌IL-6水平和T細(xì)胞分泌IL-13的水平與健康對(duì)照者相比顯著升高；EV71病毒處理健康人單核細(xì)胞和T細(xì)胞后單核細(xì)胞和T細(xì)胞分泌IL-6和IL-13能力顯著升高。結(jié)論EV71病毒可能時(shí)通過(guò)促進(jìn)單核細(xì)胞分泌IL-6和T細(xì)胞分泌IL-13，致使血清IL-6和IL-13水平升高，從而參與HFMD的發(fā)生和發(fā)展。

手足口病;白細(xì)胞介素-6；白細(xì)胞介素-13；腸道病毒71型

手足口病(hand,foot,and mouth disease，HFMD)是兒童常見(jiàn)傳染病(主要發(fā)生在5歲以下兒童)，典型的特征是口痛、厭食、低熱、手、足、口腔等部位出現(xiàn)小皰疹或小潰瘍，HFMD于1957首次在新西蘭報(bào)道[1]?？梢餒FMD的病毒包括柯薩奇病毒A16(coxsackievirus A16，CVA16)和腸道病毒71型(enterovirus 71，EV71)[2]。其中CVA16所致的HFMD并發(fā)癥發(fā)生率一般低，結(jié)局都較好[3]。而EV71所致的HFMD多出現(xiàn)較為嚴(yán)重的神經(jīng)系統(tǒng)并發(fā)癥[4-6]。已知EV71是一種具有高度嗜神經(jīng)性的正義單鏈RNA病毒，屬于小RNA病毒科，腸道病毒屬[7-9]，在一般情況下，EV71感染主要是引起兒童HFMD和皰疹性咽峽炎[10]。越來(lái)越多的證據(jù)表明，促炎和抗炎細(xì)胞因子可能在EV71所致HFMD發(fā)生中發(fā)揮關(guān)鍵的作用。目前關(guān)于白細(xì)胞介素(interleukin，IL)-6和IL-13在EV71所致HFMD發(fā)生中的作用還很少有研究報(bào)道。據(jù)此，本研究收集2013年1～12月在本院住院且確診為EV71感染的HFMD患者30例，觀察HFMD患者血清IL-6和IL-13水平變化情況及其臨床意義，并對(duì)血清IL-6和IL-13細(xì)胞來(lái)源進(jìn)行初步的探討,結(jié)果報(bào)道如下。

1 資料與方法

1.1 一般資料 根據(jù)“重癥EV71感染疾病的臨床治療專家共識(shí)”收集2013年1～12月在本院住院且確診為EV71感染的HFMD 30例，其中臨床Ⅱ期(神經(jīng)系統(tǒng)受累)10例、Ⅲ期(早期心肺衰竭)10例、Ⅳ期(心肺衰竭)10例。EV71感染HFMD的診斷標(biāo)準(zhǔn)：中國(guó)衛(wèi)生部2010年公布的 “手足口病治療指南”和“糞便EV71病毒”測(cè)試呈陽(yáng)性[11]?；颊呷朐杭闯檠@取血清，并在疾病康復(fù)時(shí)再次抽血獲取血清。同時(shí)收集同期入院健康體檢者血清10例，其中男、女各5例，年齡(26.25±11.83)歲。其中Ⅱ、Ⅲ期和Ⅳ期患者男女比例及年齡差異均無(wú)統(tǒng)計(jì)學(xué)意義，具有可比性，詳情見(jiàn)表1。

表1 3組HFMD患者臨床特征比較

1.2 酶聯(lián)免疫吸附試驗(yàn)(enzyme linked immunosorbent assay，ELISA)法檢測(cè)IL-6、IL-16 IL-6和IL-13試劑盒均購(gòu)自R&D公司，嚴(yán)格按照說(shuō)明書(shū)要求操作如下：將血清樣品和標(biāo)準(zhǔn)品100 μL加入孔中，另留出空白對(duì)照孔，用封板膜封住反應(yīng)孔，37 ℃孵育90 min；每孔加入250 μL洗滌液，震蕩1 min/次洗板，共4次；加入配制好的生物素化抗體工作液100 μL孔，37 ℃孵育60 min；洗板4次；加入酶結(jié)合物工作液100 μL孔，37 ℃孵育30 min；洗板4次；加入顯色劑100 μL/well，避光，37 ℃孵育10～20 min；加入終止液100 μL/well，短暫震蕩混勻后立即測(cè)量OD450值。

1.3 外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cell,PBMC)獲取 無(wú)菌采集的抗凝血按1∶1的比例緩緩加入已預(yù)加淋巴細(xì)胞分離液的15 mL離心管中，800×g離心15 min；滴管吸取白膜層，加入裝有10 mL無(wú)血清RPMI 1640培養(yǎng)基的15 mL離心管中，600×g離心10 min；細(xì)胞沉淀以10 mL無(wú)血清RPMI 1640培養(yǎng)基重懸，400×g離心8 min；再用1 mL RPMI-1640完全培養(yǎng)基(10 % FBS，200 IU/mL rhIL-2)將細(xì)胞沉淀重懸，臺(tái)盼藍(lán)染色計(jì)數(shù)后制備成濃度為2×106個(gè)/mL的細(xì)胞懸液。

1.4 IL-6和IL-13檢測(cè)方法 無(wú)菌分選獲取的外周血單個(gè)核細(xì)胞置于24孔板中，于24孔板中加入100 ng/mL PMA和1 μg/mL離子霉素(Ionomycin)刺激1 h，隨后加入高爾基體阻斷劑繼續(xù)孵育5 h；收集細(xì)胞，先進(jìn)性細(xì)胞表面分子CD14和CD3染色，后加入0.5 mL細(xì)胞膜固定透化液重懸細(xì)胞，室溫避光30 min；以透化液洗滌細(xì)胞2次后加入相應(yīng)熒光抗體(IL-6和IL-13)，室溫避光30 min；以透化液洗滌細(xì)胞2次，細(xì)胞重懸于0.2 mL 1%多聚甲醛固定液中待流式細(xì)胞儀檢測(cè)。

1.5 EV71感染單核細(xì)胞和T細(xì)胞方法 病毒培養(yǎng)與效價(jià)測(cè)定方法參見(jiàn)文獻(xiàn)報(bào)道[12]，EV71病毒以感染的病毒數(shù)目與細(xì)胞數(shù)目的比值(multiplicity of infection，MOI)=5的條件感染1×107的PBMC細(xì)胞。

1.6 統(tǒng)計(jì)學(xué)處理 采用SPSS16.0 軟件進(jìn)行統(tǒng)計(jì)分析，以t檢驗(yàn)或ANOVA檢驗(yàn)的方法對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析。以P<0.05為差異具有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 血清IL-6和IL-13水平分析 如圖1所示，EV71感染手足口病患者血清IL-6和IL-13水平顯著高于健康對(duì)照組(P<0.01)，且隨著疾病嚴(yán)重程度增加，血清IL-6和IL-13水平亦逐漸升高(P<0.01)。

A:血清IL-6水平檢測(cè)及其與臨床分期相關(guān)性；B:血清IL-13水平檢測(cè)及其與臨床分期相關(guān)性。

圖1 ELISA法檢測(cè)血清IL-6和IL-13水平及其與臨床分期相關(guān)性分析

2.2 治療后患者血清IL-6和IL-13水平檢測(cè) 如圖2所示，治療后患者血清IL-6和IL-13水平分別為(48.23±23.14)pg/mL和(23.25±9.63)pg/mL，與治療前的(79.63±29.45)pg/mL和(36.45±15.13)pg/mL相比顯著降低(P<0.01)。

圖2 ELISA法檢測(cè)治療后患者血清IL-6和IL-13水平

2.3 單核細(xì)胞分泌IL-6和T細(xì)胞分泌IL-13能力檢測(cè) HFMD外周血單核細(xì)胞分泌IL-6水平和T細(xì)胞分泌IL-13的水平與健康對(duì)照者相比顯著升高。

2.4 EV71病毒可誘導(dǎo)IL-6和IL-13分泌 EV71病毒處理健康人單核細(xì)胞和T細(xì)胞后單核細(xì)胞和T細(xì)胞分泌IL-6和IL-13能力顯著升高。

3 討 論

IL-6是一種重要的促炎性細(xì)胞因子，當(dāng)機(jī)體受到病原體感染或出現(xiàn)炎癥時(shí)，IL-6表達(dá)水平會(huì)出現(xiàn)顯著的升高。同時(shí)以往的研究表明，EV71感染的小鼠會(huì)出現(xiàn)短暫的IL-6、IL-10和IFN-γ水平升高[13]。EV71感染可大大增加IL-6的釋放，IL-6抗體治療后，小鼠表現(xiàn)為組織損傷程度、病毒載量和B細(xì)胞活化水平降低[14]。提示，IL-6在EV71感染HFMD發(fā)病過(guò)程中具有一定的作用。本研究結(jié)果顯示，HFMD患者血清IL-6水平顯著高于健康對(duì)照者，同時(shí)，隨著HFMD患者疾病嚴(yán)重程度增加，血清IL-6水平亦逐漸升高。該結(jié)果與Zhang等[15]通過(guò)比較99例EV71感染HFMD患者和19例健康人血清IL-6水平，發(fā)現(xiàn)的EV71感染HFMD患者血清IL-6水平顯著升高結(jié)果相一致。IL-13是一種由T細(xì)胞分泌的細(xì)胞因子，具有潛在的抗炎活性和抑制單核細(xì)胞和巨噬細(xì)胞細(xì)胞毒活性的作用[16-17]。研究顯示，EV71感染引起的肺水腫患者具有較高水平的IL-13分泌[18]。本研究結(jié)果顯示，HFMD患者血清IL-13水平顯著高于健康對(duì)照者，同時(shí)，隨著HFMD患者疾病嚴(yán)重程度增加，血清IL-13水平亦逐漸升高。提示，IL-13在EV71感染HFMD發(fā)病過(guò)程中具有一定的作用。據(jù)此，本研究的上述結(jié)果提示，IL-6和IL-13均在EV71感染HFMD發(fā)病過(guò)程中具有一定的作用。

文獻(xiàn)報(bào)道，血清中IL-6主要由單核細(xì)胞分泌，而IL-13主要由淋巴細(xì)胞分泌，據(jù)此，作者檢測(cè)了手足口病患者和健康人外周血單核細(xì)胞分泌IL-6和T細(xì)胞分泌IL-13的情況，結(jié)果發(fā)現(xiàn)，手足口病患者外周血單核細(xì)胞分泌IL-6水平和T細(xì)胞分泌IL-13的水平與健康對(duì)照相比顯著升高(P<0.01)。提示，手足口病患者血清中異常升高的IL-6和IL-13水平可能是因?yàn)閱魏思?xì)胞和T細(xì)胞分泌IL-6和IL-13能力升高所致。為了解釋單核細(xì)胞和T細(xì)胞分泌IL-6和IL-13能力升高是否為EV71病毒感染所致，本研究采用EV71病毒處理健康人單核細(xì)胞和T細(xì)胞，結(jié)果發(fā)現(xiàn)，EV71病毒可導(dǎo)致健康人單核細(xì)胞和T細(xì)胞分泌IL-6和IL-13能力升高。據(jù)此，上述結(jié)果提示，EV71病毒感染可通過(guò)上調(diào)單核細(xì)胞和T細(xì)胞分泌IL-6和IL-13的能力，進(jìn)而導(dǎo)致手足口病的發(fā)生。文獻(xiàn)報(bào)道，EV71感染后T淋巴細(xì)胞瘤Jurkat細(xì)胞分泌子IL-2、IFN-α、IL-6和IL-10的能力下降[12]，這一結(jié)果與本研究結(jié)果存在矛盾之處。推測(cè)可能的原因是本研究采用的是新鮮分離的單核細(xì)胞和T細(xì)胞，而文獻(xiàn)中采用的是淋巴瘤細(xì)胞，而瘤變的細(xì)胞功能本身就會(huì)出現(xiàn)異常的改變，同時(shí)實(shí)驗(yàn)環(huán)境的不同也會(huì)導(dǎo)致不同結(jié)果的出現(xiàn)，據(jù)此可能使得本研究結(jié)果與文獻(xiàn)報(bào)道不同。

綜述所述，本研究結(jié)果初步提示EV71病毒可能時(shí)通過(guò)促進(jìn)單核細(xì)胞分泌IL-6和T細(xì)胞分泌IL-13，致使血清IL-6和IL-13水平升高，從而參與HFMD的發(fā)生和發(fā)展。研究顯示，EV71感染后所引發(fā)的機(jī)體免疫功能紊亂、細(xì)胞炎癥因子異常級(jí)聯(lián)活化等一系列改變均與Toll樣受體(toll like receptor，TLR)密切相關(guān)[19-21]。然而，在手足口病患者中，EV71病毒感染所致單核細(xì)胞和T細(xì)胞分泌IL-6和IL-13能力升高是否與TLR相關(guān)，以及EV71病毒感染所致單核細(xì)胞和T細(xì)胞分泌IL-6和IL-13能力升高的機(jī)制還有待進(jìn)一步的深入研究。

[1]Robinson CR,Doane FW,Rhodes AJ.Report of an outbreak of febrile illness with pharyngeal lesions and exanthem:Toronto,summer 1957;isolation of group A Coxsackie virus[J].Can Med Assoc J,1958，79(3):615-621.

[2]Zhu Z,Zhu S,Guo X,et al.Retrospective seroepidemiology indicated that human enterovirus 71 and coxsackievirus A16 circulated wildly in central and southern China before large-scale outbreaks from 2008[J].Virol J,2010,7(1):300.

[3]Chang LY,Lin TY,Huang YC,et al.Comparison of enterovirus 71 and coxsackie-virus A16 clinical illnesses during the Taiwan enterovirus epidemic,1998[J].Pediatr Infect Dis J,1999,18(12):1092-1096.

[4]Chen F,Li JJ,Liu T,et al.Clinical and neuroimaging features of enterovirus71 related acute flaccid paralysis in patients with hand-foot-mouth disease[J].Asian Pac J Trop Med,2013,6(1):68-72.

[5]Chua KB,Kasri AR.Hand Foot and Mouth Disease Due to Enterovirus 71 in Malaysia[J].Virol Sin,2011,26(4):221-228.

[6]Ooi MH,Wong SC,Lewthwaite P,et al.Clinical features,diagnosis and management of human enterovirus 71 infection[J].Lancet Neurol,2010,9(11):1097-1105.

[7]Lu J,He YQ,Yi LN,et al.Viral kinetics of Enterovirus 71 in human habdomyosarcoma cells[J].World J Gastroenterol,2011,17(36):4135-4142.

[8]Yi L,He Y,Chen Y,et al.Potent inhibition of human enterovirus 71 replication by type I interferon sub-types[J].Antiviral Therapy,2011,16(1):51-58.

[9]Liao CC,Liou AT,Chang YS,et al.Immunodeficient mouse models with different disease profiles by in vivo infection with the same clinical isolate of enterovirus71 [J].J Virol,2014,88(21):12485-12499.

[10]Huang SW,Lee YP,Hung YT,et al.Exogenous interleukin-6,interleukin-13,and interferon-gamma provoke pulmonary abnormality with mild edema in enterovirus 71-infected mice[J].Respir Res,2011(12):147.

[11]蔡榕，郭嵐峰.EV71所致重癥手足口病患兒的臨床分析及其預(yù)防控制措施[J].現(xiàn)代預(yù)防醫(yī)學(xué)，2011，38(22)：4618-4619.

[12]杜伯雨，白璐，沈巖，等.腸道病毒EV71感染對(duì)T細(xì)胞免疫功能影響研究[J].醫(yī)學(xué)研究雜志，2010，39(8)：50-53.

[13]Huang SW,Lee YP,Hung YT,et al.Exogenous interleukin-6,interleukin-13,and interferon-γ provoke pulmonary abnormality with mild edema in enterovirus 71-infected mice[J].Respir Res,2011,6(12):147.

[14]Khong WX,Foo DG,Trasti SL,et al.Sustained high levels of interleukin-6 contribute to the pathogenesis of enterovirus 71 in a neonate mouse model[J].J Virol,2011,85(7):3067-3076.

[15]Zhang Y,Liu H,Wang L,et al.Comparative study of the cytokine/chemokine response in children with differing disease severity in enterovirus 71-induced hand,foot,and mouth disease[J].PLoS One,2013,8:e67430.

[16]Minty A,Chalon P,Derocq JM,et al.Interleukin-13 is a new human lymphokine regulating inflammatory and immune responses[J].Nature,1993,362(6417):248-250.

[17]De Vries JE.The role of IL-13 and its receptor in allergy and inflammatory responses[J].J Allergy Clin Immunol,1998,102(5):165-169.

[18]Chen ZF,Li RQ,Xie ZC,et al.IL-6,IL-10 and IL-13 are associated with pathogenesis in children with Enterovirus 71 infection[J].Int J Clin Exp Med,2014,7(9):2718-2723.

[19]馬慧敏，歐維琳.Toll樣受體在EV71感染發(fā)病機(jī)制中的作用[J].華夏醫(yī)學(xué)，2014，27(4)：132-135.

[20]景志忠，何小兵，房永祥，等．病毒感染對(duì)宿主TLRs模式識(shí)別與免疫應(yīng)答信號(hào)的影響[J]．病毒學(xué)報(bào)，2012，28(4)：453-461．

[21]Triantafilou K,Vakakis E,Richer EA,et al.Human rhinovirus recognition in non-immune cells is mediated by Toll-like receptors and MDA-5,which trigger a synergetic proinflammatory immune response[J].Virulence,2011,2(1):22-29.

Enterovirus 71 infection induced HFMD related to IL-6 and IL-13 level

ChenQing

(InfectionsDepartment,WuhanMedicalTreatmentCenter,Hubei430000,China)

ObjectiveTo study the clinical significance of interleukin (IL)-6 and IL-13 levels in serum of hand,foot,and mouth disease (HFMD) caused by enterovirus 71,and carries on the preliminary discussion on serum IL-6 and IL-13 cell origin.MethodsSerum levels of IL-6 and IL-13 in HFMD and healthy control were detected by enzyme linked immunosorbent assay (ELISA),serum IL-6 and IL-13 levels among different severity of HFMD were compared.Serum IL-6 and IL-13 levels in HFMD after treatment were also detected.The secretion of IL-6 by monocyte and the secretion of IL-13 by T cells in peripheral blood mononuclear cells (PBMC) were detected by Flow Cytometry (FCM).Monocyte and T cells from health individual were treated with EV71,and the secretion of IL-6 by monocyte and the secretion of IL-13 by T cells were detected by FCM too.ResultsSerum levels of IL-6 and IL-13 were (79.63±29.45) pg/mL and (36.45±15.13) pg/mL respectively in HFMD,and serum levels of IL-6 and IL-13 were (27.26±7.82) pg/mL and (13.46±3.14) pg/mL respectively in health control,serum IL-6 and IL-13 levels in HFMD were significantly higher than that of healthy subjects (P<0.01),and the mean serum IL-6 and IL-13 levels of HFMD were increased with the severity of the disease (P<0.01).After treatment,serum levels of IL-6 and IL-13 were (48.23±23.14) pg/mL and (23.25±9.63) pg/mL respectively,when compared with before treatment,serum levels of IL-6 and IL-13 were significantly decreased (P<0.01).The secretion ability of IL-6 by monocyte and the secretion of IL-13 by T cells in HFMD were significantly higher than that of healthy subjects.After infection by EV71,the secretion ability of IL-6 by monocyte and the secretion of IL-13 by T cells were significantly increased.ConclusionInfection of EV71 probably by increase the secretion ability of IL-6 by monocyte and the secretion of IL-13 by T cells,resulted in elevated of serum IL-6 and IL-13 levels,which involved in the occurrence and development of HFMD.

hand,foot and mouth disease;interleukin-6;interleukin-13;enterovirus 71

10.3969/j.issn.1671-8348.2015.26.011

陳清(1969-)，本科，副主任醫(yī)師，主要從事感染疾病方面的研究。

R725.1

A

1671-8348(2015)26-3634-03

2015-04-08

2015-06-15)