類風(fēng)濕關(guān)節(jié)炎患者外周血單核細(xì)胞亞群的變化及其意義①

錢　雷　藺　昕　陳　瑋　李　明　于月紅

(濱?？h人民醫(yī)院檢驗科，鹽城224500)

?

類風(fēng)濕關(guān)節(jié)炎患者外周血單核細(xì)胞亞群的變化及其意義①

錢雷藺昕②陳瑋李明于月紅

(濱?？h人民醫(yī)院檢驗科，鹽城224500)

目的：本次研究通過檢測類風(fēng)濕關(guān)節(jié)炎(RA)患者外周血單核細(xì)胞亞群比例及其分泌促炎細(xì)胞因子的功能，探討單核細(xì)胞亞群在RA發(fā)病過程中的作用。方法：22例RA患者(RA組)和22例健康對照者(HC組)，經(jīng)知情同意后抽取3 ml靜脈血，肝素鈉抗凝。流式細(xì)胞術(shù)(FCM)檢測單核細(xì)胞亞群比例、中間型單核細(xì)胞表面HLA-DR、Toll樣受體2(TLR2)和髓系細(xì)胞觸發(fā)受體-1(TREM-1)表達(dá)，及其細(xì)胞內(nèi)腫瘤壞死因子(TNF)-α平均熒光強(qiáng)度(MFI)，并分析RA患者單核細(xì)胞亞群比例與血清細(xì)胞因子的相關(guān)性。正態(tài)數(shù)據(jù)分析采用Students′t-test檢驗。結(jié)果：與HC組相比，RA組中間型單核細(xì)胞比例升高[(4.6±1.2)% vs (11.7±1.6)%]，差異有統(tǒng)計學(xué)意義(P<0.05)；HLA-DR表達(dá)(MFI)與HC組比較差異無統(tǒng)計學(xué)意義(26.8±8.6 vs 30.2±6.1，P>0.05)。RA組TLR2(750.2±110.3 vs 526.8±98.6)、TREM-1(58.4±12.1 vs 40.3±10.2)表達(dá)(MFI)高于HC組，差異均有統(tǒng)計學(xué)意義(P<0.05)；RA組中間型單核細(xì)胞胞內(nèi)TNF-α(46.3±6.4 vs 36.7±8.3)MFI高于HC組，差異有統(tǒng)計學(xué)意義(P<0.05)。RA患者中間型單核細(xì)胞比例與DAS28評分和血清TNF-α、白細(xì)胞介素(IL)-17呈正相關(guān)，相關(guān)系數(shù)分別為0.593(P=0.003)、0.471(P=0.027) 和0.538(P=0.009)。結(jié)論：RA患者外周血單核細(xì)胞向中間型極化，并處于活化狀態(tài)，高表達(dá)TLR2和TREM-1，分泌較多的促炎細(xì)胞因子TNF-α，參與RA的疾病過程。因此，抑制單核細(xì)胞向中間型極化或阻斷表面受體表達(dá)可能是治療RA的新途徑。

類風(fēng)濕關(guān)節(jié)炎；單核細(xì)胞亞群；Toll樣受體2；髓系細(xì)胞觸發(fā)受體-1

類風(fēng)濕關(guān)節(jié)炎(Rheumatoid arthritis，RA) 是一種以慢性多關(guān)節(jié)滑膜炎為主要特征的自身免疫性疾病。目前普遍認(rèn)為其發(fā)病機(jī)制是具有特定遺傳背景的個體，在遭受外來病原體感染的過程中，抗原提呈細(xì)胞加工處理抗原多肽，在激活T淋巴細(xì)胞產(chǎn)生抗感染效應(yīng)的同時，發(fā)生針對自身組織細(xì)胞的自身免疫應(yīng)答，引起炎性介質(zhì)增多，導(dǎo)致血管炎、滑膜增生、軟骨及骨破壞，涉及固有免疫系統(tǒng)和適應(yīng)性免疫系統(tǒng)在內(nèi)的許多不同途徑，其中包括單核細(xì)胞[1]。單核細(xì)胞通過參與病原微生物的吞噬、抗原提呈、T細(xì)胞功能調(diào)節(jié)等過程發(fā)揮重要作用。近年來，人類單核細(xì)胞分為三個亞群：經(jīng)典型(CD14highCD16-)、中間型(CD14highCD16+)和非經(jīng)典型(CD14lowCD16++)，不同的亞群具有不同的表型和功能[2]。本次研究通過檢測RA患者外周血單核細(xì)胞亞群比例及其分泌細(xì)胞因子的功能，探討單核細(xì)胞亞群在RA發(fā)病過程中的作用。

1　材料與方法

1.1材料

1.1.1研究對象本次研究經(jīng)醫(yī)院倫理委員會批準(zhǔn)(濱醫(yī)倫研2014-02)，參加者知情同意。選取2014年1月～2015年3月在濱海縣人民醫(yī)院診斷為RA患者22例(RA組)，均符合2009年美國風(fēng)濕病協(xié)會(ACR)和歐洲抗風(fēng)濕病聯(lián)盟(EULAR)修訂的RA診斷標(biāo)準(zhǔn)，用藥前采集標(biāo)本，并進(jìn)行DAS28評分。健康對照22例(HC組)，無自身免疫性疾病、腫瘤或近期感染史。各組間性別、年齡比較差異無統(tǒng)計學(xué)意義(P> 0.05)。肘前靜脈采集外周血3 ml，肝素鈉抗凝，在24 h內(nèi)完成檢測。臨床資料見表1。

1.1.2儀器和試劑異硫氰酸熒光素(FITC)鼠抗人-CD14、葉綠素蛋白偶聯(lián)物(PerCP-cy5.5)鼠抗人-CD16單克隆抗體及同型對照購自BD公司，藻紅蛋白(PE)-鼠抗人髓系細(xì)胞觸發(fā)受體-1(TREM-1)及同型對照購自R&D公司；PE-鼠抗人Toll樣受體(TLR)2、別藻藍(lán)蛋白(APC)-鼠抗人HLA-DR和PE-鼠抗人腫瘤壞死因子-α(TNF-α)及同型對照、Fixation/Permeabilization破膜/固定劑、莫能霉素、紅細(xì)胞裂解液、Perm/WashTM緩沖液購自BD公司；佛波酯(PMA)和離子霉素購自Sigma公司，流式細(xì)胞儀FACS Canto為BD公司產(chǎn)品。類風(fēng)濕因子(RF)、C反應(yīng)蛋白(CRP)采用免疫比濁法，BNProSpec特定蛋白分析儀及配套試劑檢測。液相芯片檢測儀為美國Luminex公司產(chǎn)品，血清細(xì)胞因子白細(xì)胞介素IL-6、IL-2、IL-1β、TNF-α、IL-4、IL-17檢測試劑購自Bio-Rad公司。

表1研究對象臨床資料

Tab.1Clinical characteristics of RA patients and control subjects

GroupsRApatientsHealthycontrolsPvalueAge(years)33.5±14.232.8±13.3>0.05Male/female5/175/17>0.05DAS285.7±1.80<0.05CRP(mg/L)15.78(1.2-22.6)0.26±0.12<0.05RF(U/ml)459±2410<0.05anti-CCPpositivity(%)72.7%0<0.05ESR(mm/h)35.1±12.310.1±3.3<0.05Monocytes(1×109L-1)0.39±0.140.31±0.09<0.05

1.2方法

1.2.1流式細(xì)胞術(shù)檢測外周血單核細(xì)胞亞群比例每100 μl外周血加入10 μl FITC鼠抗人-CD14、10 μl PerCP-cy5.5鼠抗人-CD16單克隆抗體、10 μl PE-鼠抗人TREM-1(或TLR2)和10 μl APC-鼠抗人HLA-DR或其同型對照，室溫避光孵育30 min，紅細(xì)胞裂解液破壞紅細(xì)胞后，磷酸鹽緩沖液(PBS)洗滌，1 500 r/min離心6 min，棄上清，400 μl PBS重懸細(xì)胞。用 FACSCanto檢測，以單核細(xì)胞設(shè)門，讀取單核細(xì)胞亞群比例、TLR2、HLA-DR和TREM-1平均熒光強(qiáng)度(MFI)或陽性細(xì)胞頻率。

1.2.2流式細(xì)胞術(shù)檢測單核細(xì)胞亞群胞內(nèi)TNF-α MFI取外周血100 μl，加入50 ng/ml PMA、1 μg/ml離子霉素和2.0 μmol/L莫能霉素，刺激培養(yǎng) 4 h，經(jīng)紅細(xì)胞裂解液破壞紅細(xì)胞后，PBS洗滌，1 500 r/min離心6 min棄上清，100 μl PBS重懸細(xì)胞。加入10 μl FITC鼠抗人-CD14、10 μl PerCP-cy5.5鼠抗人-CD16，室溫避光孵育30 min，PBS洗滌， 加入500 μl破膜/固定劑，室溫避光孵育20 min，經(jīng)Perm/WashTM緩沖液洗滌并重懸，100 μl細(xì)胞懸液中加入10 μl PE-鼠抗人TNF-α抗體，室溫避光孵育30 min。PBS洗滌后，400 μl PBS重懸細(xì)胞。以單核細(xì)胞設(shè)門，讀取單核細(xì)胞胞內(nèi)TNF-α MFI。

1.2.3液相芯片檢測血清細(xì)胞因子濃度外周血2 ml經(jīng)1 500 r/min離心5 min，收集血漿，立即儲存在-80℃冰箱。按試劑說明書檢測IL-6、IL-2、IL-1β、TNF-α、IL-4、IL-17濃度，Lumine×200讀取細(xì)胞因子濃度，每標(biāo)本測兩次，取均值。

2　結(jié)果

2.1類風(fēng)濕關(guān)節(jié)炎患者單核細(xì)胞向中間型極化外周血細(xì)胞經(jīng)流式抗體標(biāo)記后，流式細(xì)胞儀采用前向散射光(FS)和側(cè)向散射光(SS)區(qū)別單核細(xì)胞(圖1A中R1)，測定單核細(xì)胞亞群比例(圖1B：R3、R4、R5分別為非經(jīng)典型、中間型和經(jīng)典型單核細(xì)胞)。結(jié)果見表2。

RA組中間型單核細(xì)胞亞群比例高于HC組，差異有統(tǒng)計學(xué)意義(P<0.05)；經(jīng)典型單核細(xì)胞亞群比例低于HC組，差異有統(tǒng)計學(xué)意義(P<0.05)；非經(jīng)典型單核細(xì)胞亞群比例與HC組比較差異無統(tǒng)計學(xué)意義(P>0.05)。

表2單核細(xì)胞亞群比例比較

Tab.2Percentage of monocyte subpopulation comparisons

GroupsRApatientsHealthycontrolstvaluePvalueClassicalmonocytes(%)77.4±7.585.2±5.33.9830.0003Intermediatemonocytes(%)11.7±1.64.6±1.216.6510.000Nonclassicalmonocytes(%)11.2±2.110.3±2.31.3550.182

2.2中間型單核細(xì)胞表面受體表達(dá)和HC組相比，RA患者中間型單核細(xì)胞TLR2、TREM-1陽性細(xì)胞頻率差異無統(tǒng)計學(xué)意義(P>0.05)；TLR2和TREM-1MFI升高，差異有統(tǒng)計學(xué)意義(P<0.05)。HLA-DR陽性細(xì)胞頻率和MFI差異無統(tǒng)計學(xué)意義(P>0.05)。結(jié)果見圖2和表3。

2.3 RA患者中間型單核細(xì)胞胞內(nèi)高表達(dá)TNF-α外周血細(xì)胞胞內(nèi)細(xì)胞因子經(jīng)標(biāo)記后，流式細(xì)胞儀采用FS和SS區(qū)別單核細(xì)胞(圖3A中R1)，以中間型單核細(xì)胞設(shè)門(圖3B中R4)，測定單核細(xì)胞胞內(nèi)細(xì)胞因子TNF-α MFI(圖3C)。

與HC組相比，RA患者外周血中間型單核細(xì)胞胞內(nèi)TNF-α MFI表達(dá)明顯升高(46.3±6.4 vs 36.7±8.3)，差異有統(tǒng)計學(xué)意義(t= 4.296,P=0.000)。

表3中間型單核細(xì)胞表面受體表達(dá)比較

Tab.3Expression of surface receptor on interme-diate monocytes

GroupsRApatientsHealthycontrolstvaluePvalueTREM-1positivity(%)97.3±2.997.1±2.40.2490.804TREM-1MFI58.4±12.140.3±10.25.3640.000TLR2positivity(%)98.2±1.297.9±2.10.5810.563TLR2MFI750.2±110.3526.8±98.67.0820.000HLA-DRpositivity(%)13.2±4.612.9±3.80.2350.814HLA-DRMFI30.2±6.126.8±8.61.5120.137

圖1　單核細(xì)胞亞群檢測Fig.1　Analysis of monocytes subsetsNote: A.Monocytes were gated based on FS and SS;B.Monocyte subsets in healthy controls;C.Monocyte subsets in patients with RA.

圖2　中間型單核細(xì)胞亞群表面受體檢測Fig.2　Expression of cell surface molecules on intermedi-ate monocyteNote: A.Expression of monocytes TREM-1;B.Expression of monocytes HLA-DR;C.Expression of monocytes TLR2.

圖3　中間型單核細(xì)胞內(nèi)TNF-α表達(dá)Fig.3　Expression of TNF-α in intermediate monocyteNote: A.Monocytes were gated based on FS and SS;B.The subsets of intermediate monocytes;C.TNF-α MFI in intermediate monocytes.

圖4　RA患者中間型單核細(xì)胞亞群與臨床指標(biāo)相關(guān)性分析Fig.4　Relationship of certain markers with intermediate monocyte of RA patientsNote: A.Correlation of intermediate monocyte percentage and serum TNF-α;B.Correlation of intermediate monocyte percentage and DAS28；C.Correlation of intermediate monocyte percentage and IL-17.

表4血清細(xì)胞因子濃度比較(pg/ml)

Tab.4Comparison of serum cytokine concentrations

CytokineRApatientsHealthycontrolsIL-671.5(48.3-116.4)1)10.4(9.3-18.5)IL-218.3(11.4-28.1)1)11.5(8.2-18.5)IL-1β52.1(33.1-74.2)1)24.6(23.2-28.6)TNF-α31.5(14.4-62.8)1)12.4(12.3-13.8)IL-419.2(15.1-27.3)18.9(14.6-22.4)IL-1728.3(11.5-38.7)1)17.2(6.5-29.3)

Note：The skewed date were presented as medians (M,25-75 percentile).Statistical significance was determined by the Mann-WhitneyU-test,1)P<0.05 RA group vs HC group.

2.4RA患者中間型單核細(xì)胞比例與臨床數(shù)據(jù)相關(guān)性分析液相芯片檢測血清細(xì)胞因子濃度(見表4)，免疫比濁法檢測CRP、RF(見表1)，分析中間型單核細(xì)胞比例與臨床數(shù)據(jù)相關(guān)性。

RA患者中間型單核細(xì)胞比例與DAS28評分和血清TNF-α、IL-17呈正相關(guān)，相關(guān)系數(shù)分別為0.593(P=0.003)、0.471(P=0.027) 和0.538(P=0.009)，與血清IL-6、IL-4、IL-1β、CRP、IL-2濃度不相關(guān)，相關(guān)系數(shù)分別為0.031 (P=0.235)、0.012(P=0.478)、0.029 (P=0.296)、0.019(P=0.430)、0.017(P=0.515)。見圖4。

3　討論

RA是輔助性T淋巴細(xì)胞Th1/Th17為核心介導(dǎo)的免疫系統(tǒng)功能紊亂性疾病，B淋巴細(xì)胞、γδT淋巴細(xì)胞、單核細(xì)胞等免疫細(xì)胞也在疾病過程中發(fā)揮重要作用。RA患者滑膜組織有大量活化的CD4+T淋巴細(xì)胞和MHCⅡ陽性的抗原提呈細(xì)胞(APC)浸潤，其中包括單核巨噬細(xì)胞。早期的研究將單核細(xì)胞分為CD14+CD16-和CD14+CD16+兩種單核細(xì)胞亞群[3]。近年來第三類在表型和功能上具有異質(zhì)性的單核細(xì)胞亞群(CD14highCD16+) 被發(fā)現(xiàn)[4]。2010年國際免疫學(xué)會命名委員會將人類循環(huán)中單核細(xì)胞分為三種類型：經(jīng)典型(CD14highCD16-)、中間型(CD14highCD16+)和非經(jīng)典型(CD14lowCD16++)[2]，分別約占外周血單核細(xì)胞的85%、5%和10%左右。

三個亞群在表型、功能及炎癥活化潛能方面存在著明顯的差異[4]。經(jīng)典型單核細(xì)胞具有很強(qiáng)的吞噬功能；非經(jīng)典型單核細(xì)胞高表達(dá)細(xì)胞骨架能動性相關(guān)基因，能識別炎癥信號并迅速遷移到炎癥部位，浸潤組織后分化為巨噬細(xì)胞；中間型單核細(xì)胞活化后能產(chǎn)生大量促炎細(xì)胞因子，因此具有較高的活化炎癥反應(yīng)的潛能；并高表達(dá)編碼MHCⅡ類分子相關(guān)基因(如HLA-DR和CD74)，具有較強(qiáng)的抗原提呈和加工能力，促進(jìn)T細(xì)胞活化、增殖，并促進(jìn)血管生成[5]。

本次研究發(fā)現(xiàn)，與健康對照者相比，RA患者中間型單核細(xì)胞比例升高。與此研究一致的是，中間型單核細(xì)胞在子癇前期、動脈粥樣硬化、川崎病、膿毒性休克、多發(fā)性硬化癥、Ⅰ型糖尿病等多種自身免疫性和感染性疾病過程中升高，且與病情的嚴(yán)重性相關(guān)[6,7]。因此，中間型單核細(xì)胞增加可能是自身免疫性和感染性疾病的普遍現(xiàn)象，并在疾病過程中發(fā)揮重要的致病作用。

Toll 樣受體(Toll-like receptors,TLRs)是一類表達(dá)于細(xì)胞表面的模式識別受體(Pattern recognition receptor,PRR)，可識別病原相關(guān)分子模式(Pathogen-associated molecular patterns,PAMPs)。RA患者關(guān)節(jié)滑液中的成纖維細(xì)胞及巨噬細(xì)胞TLR2表達(dá)升高[8]，通過活化RA患者關(guān)節(jié)滑液中成纖維細(xì)胞TLR2，可增加TNF-α、IL-1β和IL-8、基質(zhì)金屬蛋白酶(MMPs)以及其他促炎因子的表達(dá)[9]。在RA患者病變組織中含有TLR2的內(nèi)源性配體。本次實(shí)驗證實(shí)，RA患者外周血中間型單核細(xì)胞高表達(dá)TLR2，與此研究一致的是，Iwahashi[10]早期研究表明，RA患者外周血CD14+CD16+單核細(xì)胞TLR2表達(dá)明顯上升。TREM-1是一種能激發(fā)和放大炎癥反應(yīng)的受體，經(jīng)配體活化后，促進(jìn)單核細(xì)胞分泌促炎癥細(xì)胞因子，如TNF-α、IL-1β、IL-6、粒-巨噬細(xì)胞集落刺激因子(GM-CSF)、IL-8及單核細(xì)胞趨化因子-1(MCP-1)等[11]。本次實(shí)驗證實(shí)，RA患者外周血中間型單核細(xì)胞高表達(dá)TREM-1。與此研究一致的是，RA小鼠經(jīng)編碼TREM-1細(xì)胞外結(jié)構(gòu)域和IgG-Fc的融合基因的腺病毒重組體治療后，血清炎癥因子IL-17、TNF-α、IL-1β水平顯著降低，關(guān)節(jié)面炎癥細(xì)胞浸潤減少，關(guān)節(jié)病變明顯改善[12]。通過阻斷敗血癥鼠模型TREM-1的信號轉(zhuǎn)導(dǎo)通路，不僅能減少炎癥因子的產(chǎn)生，同時不降低機(jī)體清除病原體的能力[13]。因此，TREM-1可能是RA等疾病治療的新靶點(diǎn)。

本次實(shí)驗通過胞內(nèi)染色證實(shí)，RA患者中間型單核細(xì)胞高表達(dá)TNF-α。通過分析RA患者中間型單核細(xì)胞比例與外周血細(xì)胞因子TNF-α、IL-17的相關(guān)性得到了一致的結(jié)論。TNF-α可通過誘導(dǎo)微靜脈表達(dá)細(xì)胞間黏附分子(ICAM-1)和淋巴細(xì)胞功能相關(guān)抗原(LFA)3，促使中性粒細(xì)胞、巨噬細(xì)胞聚集，從而參與RA疾病的發(fā)生發(fā)展。RA患者血清、關(guān)節(jié)滑液中TNF-α在疾病活動期升高顯著，且與RA臨床表現(xiàn)相關(guān)[14]。IL-17是一種主要由CD4+T淋巴細(xì)胞、嗜酸性粒細(xì)胞等分泌的促炎性細(xì)胞因子，具有強(qiáng)大的募集中性粒細(xì)胞及促進(jìn)多種炎性因子釋放的作用，在類風(fēng)濕關(guān)節(jié)炎的發(fā)病機(jī)制中起重要作用[15]，針對TNF-α、IL-17的生物制劑對部分患者有肯定的療效。

本次實(shí)驗發(fā)現(xiàn)中間型單核細(xì)胞比例與RA患者DAS28評分具有相關(guān)性，說明中間型單核細(xì)胞參與RA的發(fā)生發(fā)展。目前有較多研究通過減少患者體內(nèi)中間型單核細(xì)胞治療自身免疫性和感染性疾病。用吸附法去除中間型單核細(xì)胞可明顯改善泛發(fā)性膿皰性銀屑病患者的臨床癥狀[16]。研究表明，中間型單核細(xì)胞是一類比較成熟的單核細(xì)胞，可分化為樹突狀細(xì)胞(DC)，具有較強(qiáng)的抗原提呈和加工能力[17]。因此，中間型單核細(xì)胞可能是部分自身免疫性和感染性疾病的治療靶點(diǎn)。

綜上所述，本次研究表明，RA患者外周血經(jīng)典型單核細(xì)胞比例降低，中間型單核細(xì)胞比例升高，并高表達(dá)TLR2和TREM-1，分泌較多的促炎細(xì)胞因子TNF-α，參與RA的疾病過程。但RA患者中間型單核細(xì)胞極化和活化的機(jī)制尚不完全清楚。因此，調(diào)控RA患者單核細(xì)胞極化和細(xì)胞表面受體的表達(dá)可能是治療RA的新途徑。

[1]Smilek DE,Ehlers MR,Nepom GT.Restoring the balance:immunotherapeutic combinations for autoimmune disease[J].Dis Model Mech,2014,7(5):503-513.

[2]Ziegler-Heitbrock L,Ancuta P,Crowe S,etal.Nomenclature of monocytes and dendritic cells in blood[J].Blood,2010,116(16):e74-80.

[3]Passlick B,Flieger D,Ziegler-Heitbrock HW,etal.Identification and characterization of a novel monocyte subpopulation in human peripheral blood [J].Blood,1989,74(7):2527-2534.

[4]Wong KL,Tai JJ,Wong WC,etal.Gene expression profiling reveals the defining features of the classical,intermediate,and nonclassical human monocyte subsets[J].Blood,2011,118(5):e16-31.

[5]Rogacev KS,Seiler S,Zawada AM,etal.CD14(++)CD16(+) monocytes and cardiovascular outcome in patients with chronic kidney disease [J].Eur Heart J,2011,32(1):84-92.

[6]Melgert BN,Spaans F,Borghuis T,etal.Pregnancy and preeclampsia affect monocyte subsets in human and rats[J].PLos One,2012,7(9):e45229-45239.

[7]Pul R,Morbiducci F,Skuljec J,etal．Glatiramer acetate increases phagocytic activity of human monocytes in vitro and in multiple sclerosis patients[J].PLos One,2012,7(12):e51867-51878.

[8]Ospelt C,Brentano F,Jüngel A,etal.Expression,regulation,and signaling of the pattern-recognition receptor nucleotide-binding oligomerization domain 2 in rheumatoid arthritis synovial fibroblasts[J]．Arthritis Rheum，2009,60(2):355-363．

[9]Saber T，Veale DJ，Balogh E，etal.Toll-like receptor 2 induced angiogenesis and invasion is mediated through the Tie2 signalling pathway in rheumatoid arthritis [J].PLoS One,2011,6(8):e23540-23551.[10]Iwahashi M,Yamamura M,Aita T,etal.Expression of Toll-like receptor 2 on CD16+blood monocytes and synovial tissue macrophages in rheumatoid arthritis[J].Arthritis Rheum，2004,50(5):1457-1467.

[11]Qian L,Weng XW,Chen W,etal.TREM-1 as a potential therapeutic target in neonatal sepsis[J].Int J Clin Exp Med,2014,7(7):1650-1658.

[12]Tency I,Verstraelen H,Saerens B,etal.Elevated soluble triggering receptor expressed on myeloid cells (sTREM)-1 levels in maternal serum during term and preterm labor[J].PLoS One,2013,8 (2):e56050-56061.

[13]Palazzo SJ,Simpson T,Schnapp LM.Triggering receptor expressed on myeloid cells type 1 as a potential therapeutic target in sepsis[J].Dimens Crit Care Nurs,2012,31(1):1-6.

[14]Wei ST,Sun YH,Zong SH,etal.Serum levels of IL-6 and TNF-α may correlate with activity and severity of rheumatoid arthritis[J].Med Sci Monit，2015,21(4):4030-4038.

[15]Azizi G,Jadidi-Niaragh F,Mirshafiey A.Th17 Cells in Immunopathogenesis and treatment of rheumatoid arthritis[J].Int J Rheum Dis,2013,16(3):243-253.

[16]Fujisawa T,Murase K,Kanoh H,etal.Adsorptive depletion of CD14+CD16+proin ammatory monocyte phenotype in patients with generalized pustular psoriasis:clinical ef cacy and effects on cytokines[J].Ther Apher Dial,2012,16(5):436-444.

[17]Qu C,Brinck-Jensen NS,Zang M,etal.Monocyte-derived dendritic cells:targets as potent antigen presenting cells for the design of vaccines against infectious diseases[J].Int J Infect Dis,2014,19(2):1-5.

[收稿2016-01-05修回2016-03-04]

(編輯倪鵬)

Abnormality and significance of monocyte subsets in peripheral blood of patients with rheumatoid arthritis

QIAN Lei,LIN Xin，CHEN Wei,LI Ming,YU Yue-Hong.

Department of Laboratory Medicine,Binhai County People′s Hospital，Yancheng 224500，China

Objective:To explore the role of peripheral blood monocyte subsets in the pathogenesis of rheumatoid arthritis (RA),we therefore decided to compare the percentage of monocyte subpopulations in peripheral blood,as well as cytokines secretion function,to that of healthy controls.Methods: 22 patients with RA and 22 cases of healthy controls (HC) were drew 3 ml fresh venous blood into a tube containing heparin.The percentage of monocyte subsets,expression of Toll-like receptor(TLR)2,HLA-DR,triggering receptor expressed on myeloid cells-1(TREM-1) on intermediate monocyte and mean fluorescence intensity(MFI) of intracellular tumor necrosis factor-α (TNF-α) were evaluated with the methods of flow cytometry ( FCM ).The correlation between percentage of monocyte subsets and serum cytokines was explored.Statistical significance between parametric data was determined by the students′t-test.Results: Compared to HC controls,the percentages of intermediate monocytes were significant higher in RA patients [(11.7±1.6)% vs (4.6±1.2)%,P<0.05],as well as the expression(MFI) of TLR2 (750.2±110.3 vs 526.8±98.6) and TREM-1 (58.4±12.1 vs 40.3±10.2) on intermediate monocytes (P<0.05).The expression of HLA-DR on intermediate monocytes of RA patients had no difference with HC controls (P>0.05),while MFI of intracellular TNF-α in intermediate monocytes of RA patients were significant higher than that of HC controls (46.3±6.4 vs 36.7±8.3,P<0.05).In addition,RA patients showed a positive correlation between the percentage of CD14highCD16+monocytes and DAS28 scores(r=0.538,P=0.009),as well as the serum levels of TNF-α,IL-17 (r=0.471,P=0.027;r=0.593,P=0.003).Conclusion: Monocyte subpopulations from RA patients are abnormally skewed toward to intermediate monocytes which has high expression of TLR2 ,TREM-1 and the function of TNF-α secretion.Therefore,intermediate monocytes may play a role in the pathophysiology of RA.By modulating polarization or blocking monocyte cell surface receptors could be a new treatment of RA.

Rheumatoid arthritis;Monocyte subsets;Toll-like receptor 2;Triggering receptor expressed on myeloid cells-1

10.3969/j.issn.1000-484X.2016.10.023

，南京醫(yī)科大學(xué)附屬南京醫(yī)院檢驗科/南京市第一醫(yī)院，E-mail:x.lin007@163.com。

錢雷(1970年-)，男，碩士，主任技師，主要從事免疫學(xué)相關(guān)研究，E-mail:qianleiyou@163.com。

R392.8

A

1000-484X(2016)10-1519-06

①本文為鹽城市自身免疫性疾病診治創(chuàng)新團(tuán)隊項目(YCWJ-2015012)。