魏萬清 江紅 楊長(zhǎng)青 臧國(guó)慶 李丹
H BsA g抑制漿樣樹突細(xì)胞分泌IF Nα和IL-12
魏萬清 江紅 楊長(zhǎng)青 臧國(guó)慶 李丹
目的 體外研究H BsA g對(duì)漿樣樹突細(xì)胞(pD C)功能的影響。方法 磁珠分離健康人外周血pD C,CpG O D N刺激成熟,再給予H BsA g刺激,E LIS A法檢測(cè)培養(yǎng)上清中IF Nα和IL-12水平,流式細(xì)胞術(shù)檢測(cè)pD C的表面C D40、C D80、C D83和C D86抗原,激光共聚焦顯微鏡檢測(cè)pD C內(nèi)H BsA g的存在情況。結(jié)果 實(shí)驗(yàn)所需的pD C細(xì)胞純度≥95%。CpG-O D N2216刺激后,pD C表面C D40、C D80、C D83和C D86抗原的表達(dá)水平分別為78.67±2.04、83.63±3.23、71.82±2.91、70.52±2.22,培養(yǎng)上清中IF Nα和IL-12水平顯著高于對(duì)照組。上述效應(yīng)在加入H BsA g后被顯著抑制。激光共聚焦顯微鏡顯示在pD C細(xì)胞核周圍有H BsA g存在。結(jié)論 H BsA g抑制pD C的成熟和分泌功能。這種作用可能是通過pD C對(duì)H BsA g的內(nèi)吞實(shí)現(xiàn)。
乙型肝炎病毒表面抗原;漿樣樹突細(xì)胞
H B V感染的控制、轉(zhuǎn)歸及病毒清除,主要取決于宿主的免疫狀態(tài)。研究發(fā)現(xiàn),機(jī)體對(duì)H B V的耐受狀態(tài)并不是因?yàn)榱馨图?xì)胞功能缺陷,而是由于抗原提呈細(xì)胞功能缺陷,特別是樹突狀細(xì)胞(dendritic cell,D C)功能缺陷引起,而造成抗原遞呈功能障礙的原因是外界刺激不能誘導(dǎo)D C成熟,H B V本身或者H B V編碼的病毒蛋白都有可能影響抗原提呈細(xì)胞成熟[1-2]。
根據(jù)表型和功能的差別,外周血D C分為2個(gè)亞群,髓樣D C(m D C)和漿樣D C(pD C)兩類。m D C主要發(fā)揮抗原提呈作用,并分泌IL-12,誘導(dǎo)T h0細(xì)胞分化為T h1細(xì)胞,后者產(chǎn)生T h1型細(xì)胞因子介導(dǎo)細(xì)胞免疫應(yīng)答。pD C是體內(nèi)產(chǎn)生IF Nα的最主要細(xì)胞。越來越多的證據(jù)表明,pD C的數(shù)量和功能狀態(tài)與疾病的發(fā)生和預(yù)后密切相關(guān)。
H BsA g大量存在于慢性乙型肝炎患者的肝臟以及血液中,在一些患者體內(nèi)可高達(dá)100μg/m L,含量遠(yuǎn)遠(yuǎn)超過了感染性病毒顆粒[3],提示其可能在H B V拮抗宿主的免疫反應(yīng)中發(fā)揮重要的作用。本研究擬體外證實(shí)H BsA g對(duì)pD C功能的影響。
一、pD C的分離、鑒定及培養(yǎng)
抽取健康人外周血400 m L,用淋巴細(xì)胞分離液得到PB M C。應(yīng)用美天旎公司的pD C磁珠分選系統(tǒng),進(jìn)行細(xì)胞分選;將得到的細(xì)胞進(jìn)行C D123-P E及B D C A-2-FIT C抗體(B D Bioscience)標(biāo)記,應(yīng)用流式細(xì)胞儀進(jìn)行陽性細(xì)胞鑒定,證實(shí)分選得到的pD C陽性細(xì)胞純度≥95%。將分選得到的pD C分為4組,即Cp G O D N2216+H BsA g組、Cp G O D N2216組、H BsA g組和PBS組。將各組細(xì)胞在體外培養(yǎng)48 h。所有實(shí)驗(yàn)重復(fù)3次,每組實(shí)驗(yàn)設(shè)3個(gè)復(fù)孔。
二、流式細(xì)胞術(shù)檢測(cè)pD C的表面抗原
收集、洗滌并重懸4組培養(yǎng)細(xì)胞,用流式細(xì)胞儀檢測(cè)C D40、C D80、C D83和C D86的表達(dá)情況。
三、pD C培養(yǎng)上清液中IF N α和IL-12的檢測(cè)
收集4組細(xì)胞的培養(yǎng)上清液,應(yīng)用E LIS A法,檢測(cè)IF Nα(Invitrogen)和IL-12(R&D System)的表達(dá)。
四、pD C內(nèi)吞H BsA g檢測(cè)
收集4組細(xì)胞,加入0.05%TritonX-100,再加入H BsA g一抗及熒光標(biāo)記的二抗及核染料D A PI,激光共聚焦顯微鏡下觀察。
五、統(tǒng)計(jì)學(xué)分析
一、流式細(xì)胞儀檢測(cè)pD C的分離純度
只有得到高純度的pD C細(xì)胞,才使得后續(xù)實(shí)驗(yàn)順利進(jìn)行。結(jié)果如圖1所示,分選得到的pD C陽性細(xì)胞純度≥95%。
二、H BsA g抑制pD C活性分子表達(dá)及細(xì)胞因子分泌
流式細(xì)胞儀檢測(cè),新鮮分離的pD C幾乎不表達(dá)C D40、C D80、C D83和C D86等4種細(xì)胞表面抗原。Cp G-O D N2216組中的4種抗原表達(dá)百分比顯著增加,與其他3組比較,差異有統(tǒng)計(jì)學(xué)意義;Cp GO D N2216+H BsA g組中的4種抗原表達(dá)百分比,顯著低于C P G組。見表1。Cp G-O D N2216刺激pD C分泌細(xì)胞因子,這種刺激作用被H BsA g顯著抑制。見圖2。
三、pD C內(nèi)吞H BsA g
如圖3所示,pD C細(xì)胞核周圍有綠色熒光標(biāo)記的H BsA g,提示pD C內(nèi)吞H BsA g。
pD Cs是體內(nèi)產(chǎn)生IF Nα的最主要細(xì)胞,其分泌的IF N α占PB M C分泌總量的95%以上[4-5]。一旦發(fā)生病毒感染,pD C在受感染部位大量積聚,發(fā)揮強(qiáng)大的抗病毒作用[6-8]。H B V感染時(shí),亦能見到pD C在肝臟的積聚[9]。國(guó)內(nèi)外諸多研究小組關(guān)注D C的生理功能在慢性乙型肝炎(C H B)狀態(tài)下發(fā)生的改變。目前對(duì)C H B患者外周血中D C亞群的頻數(shù)是否改變還存在爭(zhēng)議,但一致認(rèn)為D C亞群的功能在C H B狀態(tài)下受損[10-12]。C H B患者外周血m D C頻數(shù)與血清A L T水平、病毒載量呈負(fù)相關(guān)[13];經(jīng)L A M抗病毒治療后,pD C數(shù)量回升[14]。
H B V感染如何導(dǎo)致D C功能受損機(jī)制并不甚清楚。一些病毒,如HIV[15]、H C V[16]等,能夠通過自身結(jié)構(gòu)蛋白抑制pD C的功能來發(fā)揮拮抗宿主免疫清除效應(yīng)。研究發(fā)現(xiàn),單核細(xì)胞源性的D C能攝取H B V抗原,但不支持H B V進(jìn)入細(xì)胞核,提示H B V不能在D C內(nèi)復(fù)制,H B V蛋白可能是干擾D C功能的因素[17]。m D C能夠吞噬H BsA g,在m D C成熟過程中如果存在H BsA g或者完整病毒顆粒導(dǎo)致m D C表面共刺激分子的表達(dá)降低,不能有效地刺激T淋巴細(xì)胞的增殖和分泌細(xì)胞因子,這些結(jié)果表明H B V完整病毒顆?;騂 BsA g具有免疫調(diào)節(jié)能力,可以直接導(dǎo)致慢性H B V感染的患者m D C功能失調(diào)[18]。這些研究結(jié)果提示,C H B患者D C的功能受損并不依賴于H B V復(fù)制,而可能與H B V的結(jié)構(gòu)蛋白相關(guān)。進(jìn)一步研究顯示,盡管H BcA g的免疫原性最強(qiáng),但在機(jī)體內(nèi)主要是通過B細(xì)胞而非D C進(jìn)行抗原提呈[19-20];且D C不能攝取外源性H BcA g,在機(jī)體內(nèi)是以H BcA g/抗-H Bc免疫復(fù)合體的形式被D C提呈[20]。研究還發(fā)現(xiàn),盡管H BcA g、不同截?cái)囿wH BcA g及H BeA g能夠在體外刺激H E K293細(xì)胞上的T L R信號(hào)通路,但H Bc/ H BeA g蛋白不是人T L R的配體,不能激活人T L R信號(hào)通路[20]。
本研究顯示,H BsA g存在條件下,pD C表面分子表達(dá)減弱,分泌IF N α和IL-12水平降低。IL-12能幫助C T L清除受感染的肝細(xì)胞,并且上調(diào)IF Nα水平。Cp G-O D N2216刺激pD C分泌IF N α和IL-12的效應(yīng)能被H BsA g抑制,提示H BsA g干擾pD C免疫功能。這種作用可能是通過pD C對(duì)H BsA g的內(nèi)吞實(shí)現(xiàn)。H BsA g抑制pD C功能的具體機(jī)制有待進(jìn)一步研究。
1 K urose K,A kbar S M,Y a m a m oto K,et al.Production of antibody to hepatitis B surface antigen (anti-H Bs)by m urine hepatitis B viruscarriers:neonatal tolerance versus antigen presentation by dendritic cells.Im m unology,1997,92:494-500.
2 Ijaz S,F(xiàn)erns R B,T edder R S.A ‘first loop’linear epitope accessible on native hepatitis B surface antigen that persists in the face of‘second loop’im m une escape.J Gen Virol,2003,84(Pt 2):269-275.
3 Patient R,Hourioux C,Sizaret P Y,et al.Hepatitis B virus subviral envelope particle m orphogenesis and intracellular trafficking.J Virol,2007,81:3842-3851.
4 Cel la M,Jarrossay D,F(xiàn)acchetti F,et al.Plasmacytoid monocytes migrate to infla med ly m ph nodes and produce large a m ounts oftype I interferon.Nat M ed,1999,5:919-923.
5 Siegal FP,Kadowaki N,Shodell M,et al.T he nature of the principal type 1 interferon-producing cells in hu man blood. Science,1999,284:1835-1837.
6 Geurtsvan Kessel C H,Willart M A,van Rijt LS,et al.Clearance of influenza virus fro m the lung depends on migratory langerin+ C D11b-but not plasmacytoid dendritic cells.J Exp M ed,2008,205:1621-1634.
7 Smit JJ,Rudd BD,Lukacs N W.Plasmacytoid dendritic cells inhibit pulm onary im m unopathology and pro m ote clearance of respiratory syncytial virus.J Exp M ed,2006,203:1153-1159.
8 L und J M,Linehan M M,Iijim a N,et al.C utting E dge:Plasmacytoid dendritic cells provide innate im m une protection against m ucosal viral infection in situ.J Im m unol,2006,177:7510-7514.
9 Zhang Z,Zou ZS,F(xiàn)u JL,et al.Severe dendritic cell perturbation is actively involved in the pathogenesis of acute-on-chronic hepatitis B liver failure.J H epatol,2008,49:396-406.
10 Beckebau m S,Cicinnati V R,D woracki G,et al.Reduction in the circulating pD C1/pD C2 ratio and im paired function of ex vivogenerated D C1 in chronic hepatitis B infection.Clin Im m unol,2002,104:138-150.
11 Arima S,A kbar S M,Michitaka K,et al.Im paired function of antigen-presenting dendritic cells in patients with chronic hepatitis B:localization of H B V D N A and H B V R N A in blood D C by in situ hybridization.Int J M ol M ed,2003,11:169-174.
12 van der M olen R G,Sprengers D,Binda R S,de Jong E C,et al. Functional im pairment of m yeloid and plasmacytoid dendritic cells of patients with chronic hepatitis B.H epatology,2004,40:738-746.
13 朱文靜,謝青,陳榕,等.C D11c+髓樣樹突狀細(xì)胞在慢性乙型肝炎中頻數(shù)與細(xì)胞表型的變化。世界華人消化雜志,2008,16:2007-2011.
14 W ang K,F(xiàn)an X,F(xiàn)an Y,et al.Study on the function of circulating plasmacytoid dendritic cells in the im m unoactive phase of patients with chronic genotype B and C H B V infection.J Viral H epat,2007,14:276-282.
15 M artinelli E,Cicala C,Van Ryk D,et al.HIV-1 gp120 inhibits T L R9-mediated activation and IF N-{alpha} secretion in plasmacytoid dendritic cells.Proc Natl Acad Sci U S A,2007,104:3396-3401.
16 Dolganiuc A,Chang S,K odys K,et al.H epatitis C virus(H C V)core protein-induced,m onocyte-mediated mechanisms of reduced IF N-alpha and plasmacytoid dendritic cell loss in chronic H C V infection.J Im m unol,2006,177:6758-6768.
17 U ntergasser A,Zedler U,Langenka m p A,et al.Dendritic cells take up viral antigens but do not support the early steps of hepatitis B virus infection.H epatology,2006,43:539-547.
18 O p den Brou w M L,Binda R S,van Roosmalen M H,Protzer U,et al.H epatitis B virus surface antigen im pairs m yeloid dendritic cell function:a possible im m une escape mechanism of hepatitis B virus.Im m unology,2009,126:280-289.
19 Milich D R,Chen M,Sch?del F,et al.Role of B cells in antigen presentation of the hepatitis B core.Proc Natl Acad Sci U S A,1997,94:14648-14653.
20 Lee B O,Tucker A,F(xiàn)relin L,Sallberg M,et al.Interaction of the hepatitis B core antigen and the innate im m une system.J Im m unol,2009,182:6670-6681.
H BsAg inhibits secretion of interferon-α and interleukin-12 from plasmacytoid dendritic cells
W EI W an-qing,JIA N G H ong,Y A N G Chang-qing,Z A N G Guo-qing,LI D an. Department ofDigestion,Tongji H ospital of Shanghai,Shanghai200065,China
Objective To investigate the effect of H BsA g on function of plasmacytoid dendritic cells(pD C)in vitro.M ethods pD Cs,sorted by im m uno magnetic beads,were divided into four groups:control group(incubated with PBS),H BsA g group(stim ulated by H BsA g),CpG-O D N group(stim ulated by CpG-O D N2216)and CpG-O D N/H BsA g group(stim ulated by CpG-O D N2216 and H BsA g).Cytokines in the cell supernatant were measured by enzy me-linked im m une sorbent assay.Surface markers were analyzed by flow cyto metry,and endocytosis of H BsA g into cytoplasma of pD Cs was imaged by confocal microscope.Results The purity of pD Cs was m ore than 95%.The freshly isolated pD Cs expressed very low levels of cell surface markers(C D40,C D80,C D83,C D86),w hich was upregulated by CpGO D N2216 but dow nregulated by H BsA g.Activated pD Cs stim ulated by CpG-O D N2216 secreted high levels ofinterferon alpha and interleukin 6,w hich could be inhibited by H BsA g.In im m unofluorescence pictures,Green H BsA g was localized in pD Cs cytoplasm and accu m ulated around cell nucleus.Conclusion H BsA g causes the defects of pD Cs im m une functions,w hich might be achieved through the endocytosis of H BsA g by pD Cs.
H epatitis B surface antigen;Plasmacytoid dendritic cells
2014-10-24)
(本文編輯:錢燕)
國(guó)家自然科學(xué)基金資助項(xiàng)目(81102279)
200001 上海市同濟(jì)醫(yī)院消化內(nèi)科(魏萬清,楊長(zhǎng)青);上海交通大學(xué)附屬第六人民醫(yī)院感染科(江紅,臧國(guó)慶,李丹)