安樂美，李 娟，季蘭嵐，李光韜，張卓莉

(北京大學(xué)第一醫(yī)院風(fēng)濕免疫科， 北京 100034)

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·論著·

類風(fēng)濕關(guān)節(jié)炎患者外周血濾泡輔助性T細(xì)胞水平檢測

安樂美，李 娟，季蘭嵐，李光韜，張卓莉△

(北京大學(xué)第一醫(yī)院風(fēng)濕免疫科， 北京 100034)

目的：檢測外周血濾泡輔助性T細(xì)胞(follicular T helper cells , Tfh cells)的比例及其表面標(biāo)志，分析與類風(fēng)濕關(guān)節(jié)炎(rheumatoid arthritis , RA)患者疾病活動(dòng)度的關(guān)系。方法： 收集40例類風(fēng)濕關(guān)節(jié)炎患者及20例正常對照組的外周血，分離外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cells, PBMC)及血清，流式細(xì)胞術(shù)檢測PBMC中CD4+CXCR5+Tfh細(xì)胞(CXCR5, 趨化因子受體, C-X-C chemokine receptor type 5)的比例及其表面程序性細(xì)胞死亡1(programmed death-1, PD-1)、可誘導(dǎo)的協(xié)同刺激性因子(inducible co-stimulator, ICOS)、CD40配體(CD40 ligand, CD40L)及白介素21受體(interleukin-21 receptor, IL-21R)的表達(dá)情況，實(shí)時(shí)熒光定量PCR方法檢測B 細(xì)胞淋巴瘤-6(B-cell lymphoma 6, Bcl-6)、白介素-21(interleukin-21, IL-21)及IL-21R的表達(dá)，酶聯(lián)免疫吸附法(enzyme-linked immunosorbent assay , ELISA)檢測血清中IL-21和B 細(xì)胞趨化因子CXC配體13 (B cell-attracting chemokine CXC ligand 13, CXCL13)水平。結(jié)果： (1)流式細(xì)胞術(shù)結(jié)果提示，與正常對照組比較，RA患者組外周血CXCR5+CD4+Tfh細(xì)胞表達(dá)與正常對照組相比明顯升高(16.75±3.92vs. 7.49±1.84，P<0.001)；低疾病活動(dòng)度或緩解組RA外周血CXCR5+CD4+Tfh細(xì)胞表達(dá)與正常對照組相比明顯升高(16.62±3.43vs. 7.49±1.84，P<0.001)；中疾病活動(dòng)度組RA外周血CXCR5+CD4+Tfh細(xì)胞表達(dá)與正常對照組相比明顯升高(16.82±3.07vs. 7.49±1.84,P<0.001)；高疾病活動(dòng)度組RA外周血CXCR5+CD4+Tfh細(xì)胞表達(dá)與正常對照組相比明顯升高(16.87±5.50vs. 7.49±1.84,P<0.001)；RA患者外周血ICOS、PD-1、IL-21R在CD4+CXCR5+細(xì)胞的表達(dá)與正常對照組相比顯著升高 (ICOS+CXCR5+CD4+細(xì)胞, 8.37±4.28vs. 3.72±1.81,P<0.001; PD-1+CXCR5+CD4+細(xì)胞, 1.57±1.10vs. 0.24±0.30,P=0.035; IL-21R+CXCR5+CD4+細(xì)胞, 4.60±4.05vs. 0.20±0.19,P=0.006)，而CD40L在CD4+CXCR5+細(xì)胞的表達(dá)在兩組間差異無統(tǒng)計(jì)學(xué)意義(3.38±3.71vs. 0.54±0.34,P=0.135)。(2)RA患者組較正常對照組外周血中IL-21R mRNA升高(5.00±4.94vs. 0.74±0.55,P<0.001)，而轉(zhuǎn)錄因子Bcl-6 mRNA [4.54(3.33, 7.23)vs. 5.31(2.81, 7.44),P=0.329]以及IL-21 mRNA [0.72(0.26, 3.45)vs. 0.56(0.27, 3.71)P=0.195]表達(dá)差異無統(tǒng)計(jì)學(xué)意義(P>0.05)；Bcl-6、IL-21和IL-21R mRNA的表達(dá)在不同組RA患者之間差異也無統(tǒng)計(jì)學(xué)意義(P>0.05)。(3)RA患者血清中IL-21、CXCL13水平明顯高于正常對照組[IL-21, (200.49±154.56) ng/Lvs. (8.21±5.95) ng/L,P<0.001; CXCL13, (43.09±1.28)ng/Lvs. (93.72±49.72) ng/L,P<0.001]；相關(guān)分析顯示IL-21、CXCL13濃度與RA疾病活動(dòng)度正相關(guān)(r>0.4)，而血清類風(fēng)濕因子(rheumatoid factor, RF)和抗環(huán)瓜氨酸多肽抗體(anti-citrullinated protein antibody, anti-CCP)與RA疾病活動(dòng)度無相關(guān)性(r<0.2)。結(jié)論： 外周血Tfh細(xì)胞及其相關(guān)細(xì)胞因子在RA患者中顯著升高，說明Tfh細(xì)胞可能參與RA的發(fā)病機(jī)制，阻斷Tfh細(xì)胞的產(chǎn)生，有望成為治療RA患者的新的作用靶點(diǎn)。

關(guān)節(jié)炎, 類風(fēng)濕；T淋巴細(xì)胞, 輔助誘導(dǎo)；細(xì)胞因子類；受體, CXCR5；白細(xì)胞介素21

類風(fēng)濕關(guān)節(jié)炎( rheumatoid arthritis, RA)是以侵襲性多關(guān)節(jié)炎為主要表現(xiàn)的異質(zhì)性、系統(tǒng)性、自身免疫性疾病，RA的基本病理變化為滑膜炎，滑膜成纖維細(xì)胞過度增生，大量T淋巴細(xì)胞和巨噬細(xì)胞浸潤以及血管翳形成[1]。RA的病因尚不清楚，可能與遺傳、感染、環(huán)境、免疫紊亂有關(guān)，其中免疫因素更重要。當(dāng)抗原進(jìn)入人體后，激活輔助性T淋巴細(xì)胞分泌細(xì)胞因子等炎性因子，激活B淋巴細(xì)胞分化為漿細(xì)胞，進(jìn)而分泌包括類風(fēng)濕因子在內(nèi)的大量免疫球蛋白，同時(shí)誘發(fā)炎癥反應(yīng)[2]。盡管RA發(fā)病機(jī)制仍未明確，但CD4+T淋巴細(xì)胞一直是研究的重點(diǎn)。濾泡輔助性T細(xì)胞(follicular helper T cells, Tfh)是新近發(fā)現(xiàn)的一種不同于Th1、Th2、Th17細(xì)胞的CD4+T細(xì)胞亞群，主要存在于次級(jí)淋巴器官濾泡中，參與生發(fā)中心的形成、維持，輔助B細(xì)胞成熟、增殖，促使免疫球蛋白發(fā)生類別轉(zhuǎn)換[3-4]。Tfh細(xì)胞特異的轉(zhuǎn)錄因子是Bcl-6，高表達(dá)趨化因子受體CXCR5(C-X-C chemokine receptor type 5, CXCR5)、程序性細(xì)胞死亡1(programmed death-1,PD-1)、可誘導(dǎo)的協(xié)同刺激性因子(inducible co-stimulator, ICOS) 等細(xì)胞表面膜分子，分泌白介素-21(interleukin-21, IL-21)等細(xì)胞因子，同時(shí)缺少Blimp-1(prdm1)[5]。外周血T細(xì)胞被認(rèn)為是外周記憶性T細(xì)胞，雖然其數(shù)量較少，但具有與Tfh細(xì)胞相似的功能特性。本文用流式細(xì)胞術(shù)檢測RA患者和正常人外周血中Tfh細(xì)胞表面標(biāo)志在CD4+細(xì)胞的表達(dá)，實(shí)時(shí)熒光定量PCR法檢測外周血IL-21、IL-21R、Bcl-6 mRNA的表達(dá)水平以及酶聯(lián)免疫吸附法測定血漿中IL-21、CXCL13水平；同時(shí)分析評價(jià)Tfh細(xì)胞相關(guān)指標(biāo)與RA疾病活動(dòng)度之間的相關(guān)性，旨在探索外周血中Tfh細(xì)胞與RA發(fā)病之間的關(guān)系，初步探討Tfh細(xì)胞在RA發(fā)病機(jī)制中的作用，并為可能的治療方法提供新策略。

1 資料與方法

1.1 資料

選取2011年7月至2013年4月就診于北京大學(xué)第一醫(yī)院風(fēng)濕免疫科的住院或門診RA患者40例，所有患者均符合1987年或2010年美國風(fēng)濕病學(xué)會(huì)(American Collage of Rheumatoid, ACR)分類標(biāo)準(zhǔn)[6-7]。排除標(biāo)準(zhǔn)：近一個(gè)月內(nèi)有感染者，近期有手術(shù)外傷史者，處于妊娠、分娩或哺乳期的女性患者，本人或直系親屬中有自身免疫性疾病，長期使用激素或免疫抑制劑，并發(fā)其他疾病者。收集正常對照20例，來自北京大學(xué)第一醫(yī)院健康職工。對照組不具備RA分類標(biāo)準(zhǔn)中的任何一條。本研究方案經(jīng)北京大學(xué)第一醫(yī)院醫(yī)學(xué)倫理委員會(huì)審批并備案(2008127)，所有入組患者和健康志愿者均簽署知情同意書。

1.2 方法

1.2.1 資料搜集 留取患者全血標(biāo)本9 mL及血清2 mL，同時(shí)收集患者的臨床資料，包括患者姓名、性別、年齡、民族、腫脹關(guān)節(jié)數(shù)(number of swollen joints, SJC)、壓痛關(guān)節(jié)數(shù)(number of tender joints, TJC)、患者對疾病的總體評分(patient global assessment of disease activity, PGA)、醫(yī)生對疾病的總體評分(evaluator globalassessment of disease activity, EGA)等臨床表現(xiàn)以及用藥情況；收集患者的實(shí)驗(yàn)室資料，包括血尿常規(guī)、紅細(xì)胞沉降率(erythrocyte sedimentation rate, ESR)、C反應(yīng)蛋白(C-reactive protein, CRP)、類風(fēng)濕因子(rheumatoid factor, RF)、抗環(huán)瓜氨酸多肽抗體(anti-citrullinated protein antibody, anti-CCP)等，為達(dá)到統(tǒng)一評判標(biāo)準(zhǔn)，臨床資料的收集均由同一醫(yī)生完成。根據(jù)上述臨床數(shù)據(jù)，計(jì)算28個(gè)關(guān)節(jié)的疾病活動(dòng)評分[the disease activity score in 28 joints, ESR-based or CRP-based, DAS28(ESR) or DAS28(CRP)]、臨床疾病活動(dòng)性指數(shù)(clinical disease activity index,CDAI)、簡化的疾病活動(dòng)指數(shù)(simplified disease activity index, SDAI)；根據(jù)DAS28(ESR)將RA患者分為3組，即低疾病活動(dòng)或緩解組、中等疾病活動(dòng)度組、高疾病活動(dòng)度組[8]。

1.2.2 實(shí)驗(yàn)方法 (1)流式細(xì)胞儀檢測:分離外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cells, PBMC)后逐級(jí)降溫凍存至液氮中，使用時(shí)復(fù)蘇凍存細(xì)胞，計(jì)數(shù)后將細(xì)胞轉(zhuǎn)移至流式管中，每管500 μL(細(xì)胞濃度維持在5×105個(gè)/mL左右)。向流式管中加入抗體CD4、CD19、PD-1、ICOS、IL-21R、CD40L及其同型對照,均為5 μL，CXCR5及其同型對照均為1.5 μL。流式1中加入CD4、CD19、 CXCR5，混勻；流式管2內(nèi)加入CD4、CD19、CXCR5相應(yīng)抗體的同型對照，混勻；流式管3中加入CD4、CXCR5、PD-1、ICOS，混勻；流式管4中加入CD4、CXCR5、PD-1、ICOS相應(yīng)抗體的同型對照，混勻；流式管5中加入CD4、CXCR5、IL-21R、CD40L，混勻；流式管6中加入CD4、CXCR5、IL-21R、CD40L相應(yīng)抗體的同型對照，混勻。室溫孵育30 min，洗滌重懸細(xì)胞，使用BD FACSAria流式細(xì)胞儀(BD公司，美國)對處理好的標(biāo)本進(jìn)行檢測，每份標(biāo)本至少獲取50 000個(gè)細(xì)胞進(jìn)行分析。用Flow Jo 7.6軟件(San Diego公司, 美國) 對獲得的數(shù)據(jù)進(jìn)行分析。(2)實(shí)時(shí)熒光定量PCR：使用Primer 3軟件設(shè)計(jì)引物，Bcl-6上游引物 5′-AAGGCCAGTGAAGCAGAGA-3′，下游引物 5′-CCGATAGGCCATGATGTCT-3′ ；IL-21上游引物 5′-TTCAGAAGGCCCAACTAAAG-3′，下游引物 5′-TGAAGGGCATGTTAGTCTGT-3′；IL-21R上游引物 5′-ACGAAGGTCTGAATCCCGA-3′，下游引物 5′-TACACGGGTGACATCGCCAA-3′；β-actin上游引物5′-CACGGCTGCTTCCAGCTC-3′，下游引物 5′-CACAGGACTCCATGCCCAG-3′。各引物委托北京天輝遠(yuǎn)有限公司合成，采取兩步法進(jìn)行實(shí)時(shí)熒光定量PCR，首先提取PBMC中的總RNA，反轉(zhuǎn)錄獲得cDNA；然后，分別分別加入IL-21，IL-21R和Bcl6的上、下游引物，同時(shí)以β-actin作為內(nèi)參照，應(yīng)用ABI 7300 RT-PCR儀，最后計(jì)算ΔCt(Ct樣本-Ct內(nèi)參照)，半定量待測目標(biāo)產(chǎn)物的表達(dá)。(3)ELISA：嚴(yán)格按照酶聯(lián)免疫吸附法(enzyme-linked immunosorbent assay, ELISA)試劑盒說明書進(jìn)行，其中所用主要試劑如下：淋巴細(xì)胞分離液購自美國GE公司；流式抗體CD4-PerCP、CD19-APC、CXCR5-Alexa Fluor?488、PD-1-APC、ICOS-PE、CD40L-PE、IL-21R-APC及與之對應(yīng)的同型對照抗體均購自美國BD PharmingenTM公司； CXCL13、IL-21 ELISA試劑盒購自美國R&D公司。

1.3 統(tǒng)計(jì)學(xué)分析

2 結(jié)果

2.1 一般臨床資料比較

RA患者與正常對照組在性別、年齡上差異無統(tǒng)計(jì)學(xué)意義，具有可比性。40例RA患者均為漢族，其中男性10例，女性30例，男女比例為1 ∶3,平均年齡(49.6±15.0)歲，病程12～75個(gè)月(中位病程30個(gè)月)，其中8例患者病程超過24個(gè)月。根據(jù)患者TJC、SJC、PGA、EGA、ESR計(jì)算DAS28(ESR)，14例患者屬于高疾病活動(dòng)度組[high disease activity of RA,HRA， DAS28(ESR)>5.1]；13例患者屬于中疾病活動(dòng)度組[moderate disease activity of RA,MRA， DAS28(ESR) 3.2～5.1]，13例患者屬于低疾病活動(dòng)度或者緩解組[low disease activity or remission of RA, LRA，DAS28(ESR)<3.2]。用藥情況見表1。20 例健康對照中，其中5例男性，15例女性，男女比例為1 ∶3.3，平均年齡 (47.8±8.8)歲，最小年齡39歲，最大年齡65歲。

表1 類風(fēng)濕關(guān)節(jié)炎患者的臨床特征(n=40)Table 1 Clinical characteristics of rheumatoid arthritis patients (n=40)

Other drugs taken were anti-tumor necrosis factor α(2 patients with active RA), hydroxychloroquine (5 patients), sulfasalazine (2 patients); some patients took>1 drug. CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; RF, rheumatoid factor; DAS28, disease activity score assessed in 28 joint areas (ESR-based or CRP-based); CDAI, clinical disease activity index; SDAI, simplified disease activity index; NSAIDs, nonsteroidal anti-inflammatory drugs.

2.2 流式細(xì)胞分析結(jié)果

2.2.1 RA患者外周血CD4+CXCR5+Tfh細(xì)胞頻率明顯升高 對RA患者及健康志愿者中的CD4+CXCR5+Tfh細(xì)胞進(jìn)行流式細(xì)胞術(shù)檢測，結(jié)果發(fā)現(xiàn)RA患者外周血CD4+CXCR5+Tfh細(xì)胞占CD4+細(xì)胞的百分比明顯高于正常對照組 (P<0.05)。進(jìn)一步對RA患者進(jìn)行分組研究，發(fā)現(xiàn)外周血CD4+CXCR5+Tfh細(xì)胞在RA患者各組間差異無統(tǒng)計(jì)學(xué)意義(圖1)。

2.2.2 CD4+CXCR5+Tfh細(xì)胞上PD-1、ICOS、CH40L和 IL-21R 的表達(dá)情況 為明確不同疾病活動(dòng)度RA患者之間及與正常對照者外周血CD4+CXCR5+Tfh細(xì)胞在細(xì)胞表型上的差異，用流式細(xì)胞術(shù)檢測了CD4+CXCR5+Tfh細(xì)胞上PD-1, ICOS, IL-21R及CD40L的表達(dá)情況，結(jié)果發(fā)現(xiàn)， RA患者中，ICOS, PD1, IL-21R在 CXCR5+CD4+細(xì)胞表達(dá)較正常對照組升高(P<0.05)，而CD40L在CXCR5+CD4+Tfh細(xì)胞的表達(dá)水平則在兩組間差異無統(tǒng)計(jì)學(xué)意義(P>0.05，圖2)。

2.3 RT-PCR實(shí)驗(yàn)結(jié)果

Bcl6和IL-21在Tfh細(xì)胞分化發(fā)育過程中起重要作用，因此本研究檢測了PBMC中相關(guān)mRNA的表達(dá)，結(jié)果顯示，IL-21R在兩組之間差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，Bcl6、IL-21 mRNA表達(dá)在RA患者和正常對照組間差異無統(tǒng)計(jì)學(xué)意義 (P>0.05，圖3)。

2.4 RA患者血清CXCL13和IL-21水平升高

血清ELISA結(jié)果示：RA組和正常對照組血漿IL-21的濃度分別為(200.49±154.56) ng/L和(8.21±5.95) ng/L，兩組間差異有統(tǒng)計(jì)學(xué)意義(P<0.001)。不同RA分組之間差異也有統(tǒng)計(jì)學(xué)意義(P<0.05)，MRA和HRA均顯著高于LRA(P<0.05)。RA組和正常對照組血漿CXCL13的濃度分別為(93.72±49.72) ng/L、(43.09±1.28) ng/L，兩組間差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。不同RA分組之間差異也有統(tǒng)計(jì)學(xué)意義(P<0.05，圖4)。

雙變量相關(guān)分析顯示：血清IL-21濃度和CXCL13濃度呈正相關(guān)(r=0.643,P<0.001)；血清CXCL13、IL-21與RA的疾病活動(dòng)指數(shù)及RF、抗CCP的相關(guān)分析見圖5和表2。

3 討論

RA是一種以進(jìn)行性滑膜炎癥和關(guān)節(jié)破壞為特征的系統(tǒng)性自身免疫病，發(fā)病率較高，是最常見的風(fēng)濕病之一。多年來，雖然對其病因及發(fā)病機(jī)制進(jìn)行了大量的研究，以尋求更有效的治療方法，但至今仍然因病因不明而使疾病無法根治。人類 Tfh 細(xì)胞是新近發(fā)現(xiàn)的一種CD4+T細(xì)胞亞群[9]，研究表明Tfh細(xì)胞參與多種自身免疫性疾病的發(fā)病，如系統(tǒng)性紅斑狼瘡、干燥綜合征、Grave’s病和橋本氏甲狀腺炎[10-12]。

本研究發(fā)現(xiàn)CXCR5+、ICOS+、PD-1+、IL-21R+細(xì)胞在CD4+細(xì)胞上表達(dá)明顯升高，與既往在系統(tǒng)性紅斑狼瘡中的研究結(jié)果一致[11]。Tfh細(xì)胞在B細(xì)胞特異性抗原發(fā)育和體液免疫反應(yīng)中起重要作用，因此增高的Tfh細(xì)胞可能反映活躍的免疫反應(yīng)[13]。本研究發(fā)現(xiàn)外周血PD-1+、ICOS+、IL-21R+在CXCR5+CD4+細(xì)胞上表達(dá)增高，說明Tfh細(xì)胞活化后從淋巴濾泡遷入至外周血發(fā)揮其功能，而外周血中CD40-CD40L依賴B細(xì)胞的活化，所以RA患者CD40L在CXCR5+CD4+細(xì)胞上表達(dá)與正常對照組間差異無統(tǒng)計(jì)學(xué)意義[14]。增多的Tfh細(xì)胞相關(guān)表面標(biāo)志與RA的疾病活動(dòng)度[ESR, CRP, DAS28(ESR), DAS28(CRP), CDAI或SDAI]沒有相關(guān)性，說明外周血Tfh細(xì)胞可能是RA的固定表型，不隨病情的變化而變化。

A, expression of CXCR5 on CD4 lymphocytes in health controls; B, expression of CXCR5 on CD4 lymphocytes in RA patients; C, expression of CXCR5 on CD4 lymphocytes between RA patients and healthy controls; D, relative prevalence of CXCR5CD4 cells among different RA groups. HC, healthy controls; RA, rheumatoid arthritis; Guided by DAS28(ESR), LRA, low disease activity or remission of RA; MRA, moderate disease activity of RA; HRA, high disease activity of RA. *P<0.001.

圖1 CXCR5CD4 Tfh細(xì)胞在RA患者與正常對照組之間的表達(dá)

Figure1 CXCR5CD4 Tfh cells expressed in RA patients and healthy controls

A, PD-1 expressed onCD4CXCR5 Tfh cells in RA patients and HC; B, ICOS expressed on CD4CXCR5 Tfh cells in RA patients and HC; C, IL-21R expressed on CD4CXCR5 Tfh cells in RA patients and HC; D, CD40L expressed on CD4CXCR5 Tfh cells in RA patients and HC. HC, healthy controls; RA, rheumatoid arthritis; PD-1, programmed death 1 positive; ICOS, inducible T cell costimulator; IL-21R, interleukin-21 receptor; CD40L, CD40 ligand. *P<0.001.

圖2 PD-1,ICOS, IL-21R和CD40L在CD4CXCR5 Tfh細(xì)胞表達(dá)

Figure2 PD-1, ICOS, IL-21R and CD40L expressed on CD4CXCR5 Tfh cells in RA patients and HC

A, Bcl-6 mRNA expressed in RA and HC; B, IL-21 mRNA expressed in RA and HC; C, IL-21R mRNA expressed in RA and HC; Bcl-6, B-cell lymphoma 6; IL-21, Interleukin-21; IL-21R, Interleukin-21 receptor; HC, healthy controls; RA, rheumatoid arthritis. * P<0.001.

圖3 RA患者與正常對照組之間Bcl-6、IL-21和IL-21R mRNA的表達(dá)

Figure3 Expression of Bcl-6, IL-21 and IL-21R mRNA

A, level of CXCL13 in sera of RA patients and HC;B, level of CXCL13 in different RA subgroups; C, level of IL-21 in sera of RA patients and HC; D, level of IL-21 in different RA subgroups; HC, healthy controls; RA, rheumatoid arthritis. IL-21, Interleukin-21; CXCL13,B cell-attracting chemokine CXC ligand 13; LRA, low disease activity or remission of RA; MRA, moderate disease activity of RA; HRA, high disease activity of RA. *P<0.001.

圖4 CXCL13、IL-21在RA患者和正常對照組血清中的表達(dá)

Figure4 CXCL13 and IL-21 levels in sera of RA patients and HC

A, relationship between serum CXCL13 and ESR; B, relationship between serum CXCL13 and DAS28(ESR); C, relationship between serum CXCL13 and DAS28(CRP); D, relationship between serum CXCL13 and CDAI; E, relationship between serum CXCL13 and SDAI; F, relationship between serum CXCL13 and IL-21. ESR, erythrocyte sedimentation rate; CRP, C reactive protein; DAS28(ESR) or DAS28(CRP), the disease activity score in 28 joints, ESR-based or CRP-based; CDAI, clinical disease activity index; SDAI, simplified disease activity index; IL-21, Interleukin-21; CXCL13,B cell-attracting chemokine CXC ligand 13.圖5 血清CXCL13濃度與RA活動(dòng)度指標(biāo)[ESR、DAS 28(ESR)、DAS 28(CRP)、CDAI、SDAI]及血清IL-21濃度

Figure5 Relationship between serum CXCL13 and RA disease activity indexes and serum IL-21 in RA patients

表2 細(xì)胞因子濃度與疾病活動(dòng)度指標(biāo)、自身抗體定量資料的相關(guān)性分析

ESR, erythrocyte sedimentation rate; CRP, C reactive protein; DAS28(ESR) or DAS28(CRP), the disease activity score in 28 joints, ESR-based or CRP-based;CDAI, clinical disease activityindex; SDAI, simplified disease activity index; IL-21, Interleukin-21; CXCL13,B cell-attracting chemokine CXC ligand 13; RF, rheumatoid factor; anti-CCP, anti-citrullinated protein antibody.

RA患者和正常對照組之間轉(zhuǎn)錄因子Bcl-6的表達(dá)差異無統(tǒng)計(jì)學(xué)意義，分析原因可能是 Bcl-6的表達(dá)為轉(zhuǎn)錄后調(diào)控，檢測Bcl-6 蛋白比Bcl-6 mRAN更能反映問題的本質(zhì)[15]。此外，IL-21 mRNA的表達(dá)在RA患者和正常對照組之間差異無統(tǒng)計(jì)學(xué)意義，說明IL-21也可在其他組織中表達(dá)，如滑膜。IL-21主要由活化的CD4+T細(xì)胞分泌，所以IL-21可能在活動(dòng)度非常高的RA患者中表達(dá)升高[16]。

本研究發(fā)現(xiàn)RA患者中血漿CXCL13升高，且與RA患者的病情活動(dòng)度正相關(guān)，這促使我們檢測CXCL13的受體CXCR5的表達(dá)。外周血中的CXCR5+細(xì)胞群增多，是否與 CXCL13的趨化作用有關(guān)，需要進(jìn)一步驗(yàn)證，而在SLE中，CXCL13與系統(tǒng)性紅斑狼瘡疾病活動(dòng)指數(shù)相關(guān)[17]。血漿CXCL13與CD4+CXCR5+之間的相關(guān)性說明CXCL13可作為代替CD4+CXCR5+的評價(jià)指標(biāo)。

本研究發(fā)現(xiàn)RA患者中循環(huán)Tfh細(xì)胞表達(dá)增高，然而改善病情抗風(fēng)濕藥治療后是否降低Tfh表達(dá)的相關(guān)表達(dá)尚不能確定。如果Tfh細(xì)胞異常，可能在不同程度上影響T細(xì)胞B細(xì)胞的相互作用，導(dǎo)致體液免疫和細(xì)胞免疫的異常，從而導(dǎo)致RA發(fā)病。

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(2015-02-05收稿)

(本文編輯：劉淑萍)

Detection of peripheral follicular helper T cells in rheumatoid arthritis

AN Le-mei, LI Juan, JI Lan-lan, LI Guang-tao, ZHANG Zhuo-li△

(Department of Rheumatology and Clinical Immunology, Peking University First Hospital, Beijing 100034, China)

Objective： To detect cell frequency and surface markers of peripheral CD4+CXCR5+follicular helper T (Tfh) cells and analyze the correlation between CD4+CXCR5+Tfh cells and rheumatoid arthritis (RA) disease activity. Methods: Forty RA patients meeting the American College of Rheumatology classification criteria for RA and twenty healthy controls (HC) were included. The peripheral blood mononuclear cells and sera were collected. The expressions of CD4+CXCR5+Tfh cells (CXCR5, C-X-C chemokine receptor type 5) and inducible T cell costimulator (ICOS), programmed death 1 positive (PD-1), interleukin-21 receptor (IL-21R) and CD40 ligand (CD40L) positive on CD4+CXCR5+Tfh cells were analyzed by flow cytometry. The transcript levels of B-cell lymphoma 6 (Bcl-6), as well as IL-21 and IL-21R, were measured by real-time polymerase chain reaction. Besides, serum IL-21 and CXCL13 concentrations were determined by enzyme-linked immunosorbent assay. The potential association between Tfh cells and RA disease activity was detected. Results: The cell surface marker of CXCR5+on CD4+cells was significantly increasingly higher across the following groups versus HC: total RA patients (16.75±3.92vs.7.49±1.84,P<0.001); RA patients with low disease activity or remission (16.62±3.43vs. 7.49±1.84,P<0.001); RA patients with moderate disease activity (16.82±3.07vs. 7.49±1.84,P<0.001) and RA patients with high disease activity (16.87±5.50vs. 7.49±1.84,P<0.001). Besides, the percentages of ICOS+, PD-1+, IL-21R+on CD4+CXCR5+Tfh cells in the RA patients were significantly higher than that of HC (ICOS+CD4+CXCR5+cells, 8.37±4.28vs. 3.72±1.81,P<0.001; PD-1+CD4+CXCR5+cells, 1.57±1.10vs. 0.24±0.30,P=0.035; IL-21R+CD4+CXCR5+cells, 4.60±4.05vs. 0.20±0.19,P=0.006). But the percentage of CD40L+on CXCR5+CD4+Tfh cells in the RA patients was not significantly higher than that of HC (3.38±3.71vs. 0.54±0.34,P=0.135). The IL-21R mRNA expression was elevated significantly (5.00±4.94vs. 0.74±0.55,P<0.001) in the RA patients but not in Bcl-6 mRNA[4.54(3.33, 7.23)vs. 5.31(2.81, 7.44),P=0.329]or IL-21 mRAN[0.72(0.26, 3.45)vs. 0.56(0.27, 3.71),P=0.195]. Additionally, the serum interleukin-21 (IL-21)and CXCL13 levels in the RA patients were higher than in the healthy controls [IL-21, (200.49±154.56) ng/Lvs. (8.21±5.95) ng/L,P<0.001; CXCL13, (93.72±49.72) ng/Lvs. (43.09±1.28) ng/L,P<0.001] and were both positively correlated with RA disease activity indexes, including erythrocyte sedimentation rate, the disease activity score in 28 joints (ESR-based or CRP-based), clinical disease activity index, and simplified disease activity index. However, none of the Tfh cells, anti-citrullinated protein antibody or rheumatoid factor had any relationship with RA disease activity. Conclusion: Peripheral Tfh cells and their relevant cytokines are higher in RA patients than healthy controls, indicating Tfh cells may participate in the pathogenesis of RA, therefore, blocking the pathway of Tfh cells may be reasonable cellular targets for therapeutic intervention.

Arthritis, rheumatoid; T-lymphocytes, helper-inducer; Cytokines; Receptors, CXCR5; Interleukin-21

國家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃(973計(jì)劃，2010CB529103)資助Supported by the Major State Basic Research Development Program of People’s Republic of China (973 Program, 2010CB529103)

時(shí)間：2016-10-31 16：28：45

http://www.cnki.net/kcms/detail/11.4691.R.20161031.1628.012.html

R593.22

A

1671-167X(2016)06-0951-07

10.3969/j.issn.1671-167X.2016.06.006

△ Corresponding author’s e-mail, zhuoli.zhang@126.com