中国医科大学学报  2018, Vol. 47 Issue (6): 556-561

文章信息

范晓蕾, 刘中华, 岳涛, 何东仪
FAN Xiaolei, LIU Zhonghua, YUE Tao, HE Dongyi
血清TNF-α、IL-1β、IL-6和IL-17表达水平在阿达木单抗治疗活动性类风湿关节炎中的疗效预测作用
Changes in Serum Tumor Necrosis Factor-α, Interleukin-1β, Interleukin-6, and Interleukin-17 Levels and Their Predictive Value in Clinical Responses to Adalimumab Treatment in Patients with Rheumatoid Arthritis
中国医科大学学报, 2018, 47(6): 556-561
Journal of China Medical University, 2018, 47(6): 556-561

文章历史

收稿日期:2017-06-09
网络出版时间:2018-05-21 14:43
血清TNF-α、IL-1β、IL-6和IL-17表达水平在阿达木单抗治疗活动性类风湿关节炎中的疗效预测作用
范晓蕾1 , 刘中华2 , 岳涛1 , 何东仪1     
1. 上海市光华中西医结合医院关节内科, 上海 200052;
2. 复旦大学附属中山医院肾内科, 上海 200032
摘要目的 评估活动性类风湿关节炎(RA)在阿达木单抗(ADA)治疗前后患者血清促炎细胞因子表达水平的变化及其疗效预测作用。方法 本研究共连续纳入79例活动性RA患者。所有患者均接受ADA 40 mg/2周治疗,持续治疗12周。于基线期和治疗12周后采集血清标本,并采用ELISA检测肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-6和IL-17的表达水平。结果 ADA治疗12周后,患者C反应蛋白(CRP)、血沉(ESR)、28关节疾病活动评分(DAS28)相对于基线期均显著下降(均P < 0.001),同时血清TNF-α(P < 0.001)、IL-1β(P=0.007)、IL-6(P < 0.001)和IL-17(P < 0.001)水平也显著降低。基线期应答组患者IL-1β(P=0.088)和IL-6(P=0.092)水平数值上高于未应答组,单因素逻辑回归分析发现,基线期IL-1β高表达(P=0.091)和IL-6高表达(P=0.062)可在一定程度上预测临床应答,而多因素逻辑回归分析后发现它们均无独立预测作用。结论 ADA治疗可以显著改善RA患者疾病活动度,下调血清TNF-α、IL-1β、IL-6和IL-17表达水平,此外,治疗前血清IL-1β和IL-6水平可能在一定程度上预测ADA治疗RA患者的临床应答。
关键词阿达木单抗    类风湿关节炎    临床应答    炎性细胞因子    
Changes in Serum Tumor Necrosis Factor-α, Interleukin-1β, Interleukin-6, and Interleukin-17 Levels and Their Predictive Value in Clinical Responses to Adalimumab Treatment in Patients with Rheumatoid Arthritis
1. Department of Arthrology, Guanghua Integrative Medicine Hospital, Shanghai 200052, China;
2. Department of Nephrology, Zhongshan Hospital Affiliated to Fudan University, Shanghai 200032, China
Abstract: Objective To investigate the changes in serum levels of pro-inflammatory cytokines and their predictive value in clinical responses to adalimumab(ADA) treatment in patients with rheumatoid arthritis(RA). Methods This study consecutively enrolled 79 active RA patients who received 40 mg ADA every 2-weeks for 12 weeks. Serum samples were obtained at the baseline and at 12 weeks post treatment. Serum tumor necrosis factor(TNF) α, interleukin(IL) -1β, IL-6, and IL-17 levels were detected by ELISA. Results CRP, ESR, and the DAS28 score decreased after 12-week ADA treatments(all P < 0.001). Serum TNF-α(P < 0.001), IL-1β(P=0.007), IL-6 (P < 0.001), and IL-17(P < 0.001) levels also decreased. The baseline IL-1β(P=0.088) and IL-6(P=0.092) levels were found to be elevated in the response group compared to that in the non-response group. Univariate logistic regression analysis revealed that high baseline expression of IL-1β(P=0.091) and IL-6(P=0.062) might predict a clinical response to ADA treatment; however, multivariate logistic regression analysis revealed that neither IL-1β nor IL-6 could predict clinical response independently. Conclusion Serum TNF-α, IL-1β, IL-6, and IL-17 levels were downregulated in patients with RA after ADA therapy, and high baseline expression of IL-1β and IL-6 might predict clinical response to ADA treatment.

类风湿关节炎(rheumatoid arthritis,RA)是一种常见的慢性自身免疫性疾病,以滑膜炎症、关节炎症及软骨破坏为主要临床表现,发病后若未及时治疗,40%~70%的患者最终会发展为关节畸形、功能障碍,甚至残疾[1-2]。此外,RA患者还常并发间质性肺病、心血管疾病等关节外疾病,给患者和社会均带来沉重的疾病和经济负担[3-5]。阿达木单抗(adalimumab,ADA),作为一种新的全人源肿瘤坏死因子(tumor necrosis factor,TNF)单克隆抗体,可以快速缓解RA患者炎症和疾病活动度,并且在一定程度上抑制放射学进展[6-8]。尽管如此,依然约有20%~30%的RA患者对于ADA治疗应答不佳或难以承受其高昂的价格,因此积极探寻ADA治疗RA患者的新颖、可信的疾病监控和疗效预测标志物十分必要。TNF-α、白细胞介素1β(interleukin,IL-1β)、IL-6和IL-17,作为重要的炎性细胞因子,在RA的疾病发生和发展中均起着至关重要的作用。本研究拟评估活动性RA患者血清TNF-α、IL-1β、IL-6和IL-17表达水平在ADA治疗前后的变化及其疗效预测作用。

1 材料与方法 1.1 研究对象

本研究前瞻性队列研究,共连续纳入79例于2013年1月至2014年12月在上海市光华中西医结合医院就诊的活动性RA患者。纳入标准为:(1)符合1987年美国风湿病学会(american college of rheumatology,ACR)的RA分类标准或2010年ACR/欧洲抗风湿病联盟(european league against rheumatism,EULAR)的RA分类标准;(2)年龄18~65周岁;(3)同时符合以下内容中A、B以及C、D、E 3项中任一项的中高活动度RA患者,A,关节肿胀≥4个;B,关节压痛≥6个;C,晨僵持续时间≥45 min;D,血沉(魏氏法)≥28 mm/h;E,C反应蛋白(c-reaction protein,CRP) > 10 mg/L;(4)准备使用ADA进行治疗。排除标准:(1)6个月内接受过生物制剂的治疗;(2)合并血液恶性疾病或其他恶性实体肿瘤;(3)合并中度以上肝功能或肾功能不全;(4)有严重感染史;(5)难以正常随访。本研究获得了上海市光华中西医结合医院伦理委员会的批准,所有患者均签署了知情同意书。

1.2 治疗和信息采集

所有RA患者均按照意愿和临床实际需求接受ADA 40 mg/2周治疗,持续12周。基线期记录患者人口学和临床信息,包括:年龄、性别、病程、类风湿因子(rheumatoid factor,RF)、抗瓜氨酸化蛋白抗体(anticitrullinated protein antibodie,ACPA)、CRP、红细胞沉降率(erythrocyte sedimentation rate,ESR)和28关节疾病活动评分(disease activity score in 28 joints,DAS28)。治疗12周后,再次记录患者CRP、ESR和DAS28评分。临床应答采用EULAR治疗应答标准进行评估,即DAS28下降值 > 1.2分。

1.3 血样采集和炎性细胞因子检测

于基线期和治疗12周后分别采集RA患者4 mL外周血,并立刻分离血清置于-80℃冰箱保存。采用酶联免疫吸附测定实验(enzyme linked immunosorbent assay,ELISA)试剂盒(美国eBioscience公司)检测血清TNF-α、IL-1β、IL-6和IL-17的表达水平。

1.4 统计学分析

采用SPSS 22.0软件进行统计分析。数据以x±s、中位数(25~75分位值)或频数(百分比)表示。2组间比较采用t检验或Wilcoxon秩和检验,同组治疗前后比较采用Wilcoxon符号秩和检验。采用单因素逻辑回归模型分析ADA治疗RA患者临床应答的预测因素,所有单因素模型中P ≤ 0.1的因素进一步采用多因素逻辑回归模型进行分析。P < 0.05为差异有统计学意义。

2 结果 2.1 一般信息

79例入选RA患者中,7例患者未完成研究,其中4例失访,2例患者自愿退出,1例出现不良反应(肝功能异常,予以停药和保肝处理),因此最终共72例患者纳入分析。72例患者平均年龄为(48.3±12.4)岁,69%为女性,中位病程为71(32~93)个月,平均DAS28评分为5.8±1.4。55例(76%)患者为RF阳性,52例(72%)患者为ACPA阳性。见表 1

表 1 RA患者基线期信息 Tab.1 Baseline characteristics of RA patients
Parameter RA patients(n = 72)
Age(year) 48.3±12.4
Female [n(%)] 50(69)
Disease duration(month) 71(32-93)
ESR(mm/h) 35.0(15.4-46.2)
CRP(mg/L) 28.7(19.9-38.9)
DAS28 score 5.8±1.4
RF positive [n(%)] 55(76)
ACPA positive [n(%)] 52(72)
Concomitant medications [n(%)]
 MTX 30(42)
 LEF 32(44)
 SSZ 10(14)
 HCQ 12(17)
ESR,erythrocyte sedimentation rate;CRP,C-reactive protein;DAS28,disease activity score in 28 joints;RF,rheumatoid factor;ACPA,anticitrullinated protein antibodies;MTX,methotrexate;LEF,leflunomide;SSZ,sulfasalazine;HCQ,hydroxychloroquine.

2.2 治疗疗效

ADA治疗12周后炎症水平显著降低,ESR由35.0(27.5~46.2)mm/h下降至18.7(12.5~26.8)mm/h(P < 0.001,图 1A);CRP由28.7(19.9~38.9)mg/L降低至14.6(9.5~22.3)mg/L(P < 0.001,图 1B)。同时,ADA还显著降低了疾病活动度,DAS28评分由5.8±1.4下降至3.3±0.9(P < 0.001,图 1C)。按照EULAR治疗应答定义,共有45例患者(62.5%)达到了临床应答,27例患者(37.5%)未达到临床应答。

A, ESR; B, CRP; C, DAS28 score.* P < 0.001 vs baseline. 图 1 ADA治疗12周后ESR、CRP和DAS28评分显著下降 Fig.1 EST, CRP, and DAS 28 scores markedly decreased after 12 weeks of treatment with ADA

2.3 治疗前后血清TNF-α、IL-1β、IL-6和IL-17水平的变化

ADA治疗12周后,RA患者血清TNF-α(P < 0.001)、IL-1β(P = 0.007)、IL-6(P < 0.001)和IL-17(P < 0.001)表达水平均较基线期显著降低(表 2)。将患者分为应答组和未应答组评估时发现,应答组患者血清TNF-α(P < 0.001)、IL-1β(P < 0.001)、IL-6(P < 0.001)和IL-17(P < 0.001)表达水平相对于基线显著下降,而未应答患者仅血清TNF-α(P = 0.036)水平有所下降。

表 2 治疗前后TNF-α、IL-1β、IL-6和IL-17的变化(pg/mL) Tab.2 Changes in TNF-α, IL-1β, IL-6, and IL-17 levels after treatment(pg/mL)
Patients TNF-α IL-1β IL-6 IL-17
All patients(n = 72)
  Baseline 53.522(32.437-109.679) 3.431(2.397-4.828) 42.524(21.241-94.135) 50.140(36.221-84.957)
  12 weeks 28.651(19.429-45.391) 2.510(1.398-3.672) 26.381(18.365-46.387) 32.784(23.855-53.840)
  P < 0.001 0.007 < 0.001 < 0.001
Responders(n = 45)
  Baseline 60.410(32.996-112.316) 3.617(2.611-5.108) 46.919(23.658-102.342) 52.065(39.410-85.964)
  12 weeks 26.382(15.339-42.914) 2.626(1.260-3.963) 26.882(19.384-44.381) 27.380(22.625-52.158)
  P < 0.001 < 0.001 < 0.001 < 0.001
Non-responders(n = 27)
  Baseline 49.076(27.219-84.686) 2.681(2.348-3.857) 29.060(19.638-67.820) 48.214(23.505-83.244)
  12 weeks 34.827(26.350-64.392) 2.198(1.683-3.106) 24.843(17.382-54.283) 38.371(26.380-54.627)
  P 0.036 0.142 0.251 0.195

2.4 基线血清TNF-α、IL-1β、IL-6和IL-17水平与临床应答的关联

基线期应答组患者血清IL-1β(P = 0.088)和IL-6(P = 0.092)水平高于未应答组,但是差异无统计学意义。2组基线期TNF-α(P = 0.446)和IL-17(P = 0.242)水平未见差异,见图 2

A, TNF-α levels; B, IL-1β levels; C, IL-6 levels; D, IL-17 levels. 图 2 应答组和未应答组TNFα、IL-1β、IL-6和IL-17表达水平 Fig.2 Levels of TNFα, IL-1β, IL-6, and IL-17 in responders and non-responders

2.5 预测临床应答基线期因素分析

单因素逻辑回归分析显示,基线期IL-1β高表达(P = 0.091)、IL-6高表达(P = 0.062)和高CRP水平(P = 0.091)可能在一定程度上可以预测ADA治疗RA患者12周后的临床应答。单因素回归中所有P≤0.1的因素进一步纳入多因素逻辑回归分析,结果显示,IL-1β高表达(P = 0.338)、IL-6高表达(P = 0.067)均不是临床应答的独立预测因素,仅高CRP水平可以独立预测临床应答(P = 0.023),见表 3

表 3 临床应答的预测因素分析 Tab.3 Predictive analysis of clinical response
Item Univariate logistic regression model Multivariate logistic regression model
P OR 95% CI P OR 95% CI
Lower Higher Lower Higher
TNF-α high expression 0.226 1.818 0.691 4.782 - - - -
IL-1β high expression 0.091 2.326 0.873 6.197 0.338 1.694 0.576 4.985
IL-6 high expression 0.062 2.550 0.954 6.813 0.067 3.014 0.927 9.800
IL-17 high expression 0.808 1.126 0.433 2.925 - - - -
Age > 49 years 0.808 0.888 0.342 2.308 - - - -
Gender(female) 0.238 0.518 0.174 1.546 - - - -
Disease duration > 71 months 0.362 0.640 0.245 1.672 - - - -
ESR > 35.0 mm/h 0.466 1.429 0.548 3.726 - - - -
CRP > 28.7 mg/L 0.091 2.326 0.873 6.197 0.023 3.686 1.193 11.384
DAS28 > 5.6 0.177 1.952 0.739 5.153 - - - -
RF(positive) 0.354 1.684 0.559 5.073 - - - -
ACPA(positive) 0.178 2.059 0.720 5.888 - - - -
MTX 0.538 1.360 0.512 3.615 - - - -
LEF 0.624 1.273 0.485 3.343 - - - -
SSZ 0.383 0.550 0.143 2.110 - - - -
HCQ 0.744 1.243 0.336 4.599 - - - -
ESR,erythrocyte sedimentation rate;CRP,C-reactive protein;DAS28,disease activity score in 28 joints;RF,rheumatoid factor;ACPA,anticitrullinated protein antibodies;MTX,methotrexate;LEF,leflunomide;SSZ,sulfasalazine;HCQ,hydroxychloroquine.

3 讨论

ADA是目前全球使用最广泛的TNF抑制剂,越来越多的证据表明,ADA治疗活动性RA患者可以获得快速的疾病活动度下降以及炎症控制。KAVANAUGH等[9]纳入了大样本(1 032例)中高活动度早期RA患者,发现ADA治疗12周即可以快速降低患者CRP水平和DAS28评分,持续治疗至26周可获得高达44%的低疾病活动度缓解。ENG等[10]研究表明,针对长病程中高活动度RA患者,ADA治疗6个月降低了患者ESR水平、CRP水平和DAS28评分。与之前研究结果一致,本研究发现ADA治疗中高活动度RA患者,可以快速下调ESR和CRP水平,并且降低DAS28评分,这是由于ADA快速强效结合TNF-α,下调了炎症细胞和炎性因子水平,从而降低了炎症和疾病活动度。

YUE等[11]通过一项队列研究发现,ADA治疗RA患者12周显著下调了外周血CD4(+)IL-17(+)细胞比例,并同时降低了血清IL-6和IL-17的表达水平。AERTS等[12]研究表明,ADA治疗RA患者显著降低了其外周血TNF-α和IL-1β的表达水平。与上述研究一致,本研究发现,ADA治疗RA患者可以快速控制炎症,显著下调血清TNF-α、IL-1β、IL-6和IL-17表达水平,这和ADA的强效抑炎作用有关。此外,本研究还发现ADA治疗后应答组患者TNF-α、IL-1β、IL-6和IL-17表达水平均显著降低,而未应答组仅有TNF-α水平有所下降,其余IL-1β、IL-6和IL-17表达水平相对于基线均未有显著变化,类似研究少见报道,这可能是由于未应答患者治疗后依然处在较高的疾病活动度(DAS28改变 < 1.2分),因此炎症细胞和炎性因子依然较高水平表达。此外,本研究还发现IL-1β和IL-6在一定程度上可以预测ADA治疗RA患者的临床应答。这可能是由于IL-1β和IL-6高表达与基线期高度炎症相关,而临床应答是由指标相对于基线变化差值决定的,因此高度的炎症可能提供更大的炎症降低阈值空间,进而更可能达到临床应答。此外,IL-1β和IL-6水平可能和抗抗体的产生相关,越高水平的炎性环境越少可能出现抗抗体,进而更可能实现应答。

本研究尚存在一些缺陷:(1)样本量较少,因此缺乏较高的统计效能;(2)研究随访期过短(12周),因此血清TNF-α、IL-1β、IL-6和IL-17水平在ADA长期治疗中的应用未达到评估。

综上所述,ADA治疗RA患者可以显著改善其疾病活动度,下调血清TNF-α、IL-1β、IL-6和IL-17表达水平,此外,治疗前血清IL-1β和IL-6水平可能在一定程度上可以预测ADA治疗RA患者的临床应答。

参考文献
[1]
CATRINA AI, SVENSSON CI, MALMSTROM V, et al. Mechanisms leading from systemic autoimmunity to joint-specific disease in rheumatoid arthritis[J]. Nat Rev Rheumatol, 2017, 13(2): 79-86. DOI:10.1038/nrrheum.2016.200
[2]
SMOLEN JS, ALETAHA D, MCLNNES IB. Rheumatoid arthritis[J]. Lancet, 2016, 4(3): 427-442. DOI:10.1016/S0140-6736(16)30173-8
[3]
RADNER H, LESPERANCE T, ACCORTT NA, et al. Incidence and prevalence of cardiovascular risk factors among patients with rheumatoid arthritis, psoriasis, or psoriatic arthritis[J]. Arthritis Care Res (Hoboken), 2016, 69(10): 1510-1518. DOI:10.1002/acr.23171
[4]
ZAMORA-LEGOFF JA, KRAUSE ML, CROWSON CS, et al. Patterns of interstitial lung disease and mortality in rheumatoid arthritis[J]. Rheumatology(Oxford), 2016, 56(3): 344-350. DOI:10.1093/rheumatology/kew391
[5]
TAYLOR PC, MOORE A, VASILESCU R, et al. A structured literature review of the burden of illness and unmet needs in patients with rheumatoid arthritis:a current perspective[J]. Rheumatol Int, 2016, 36(5): 685-695. DOI:10.1007/s00296-015-3415-x
[6]
BURMESTER GR, MATUCCI-CERINIC M, MARIETTE X, et al. Safety and effectiveness of adalimumab in patients with rheumatoid arthritis over 5 years of therapy in a phase 3b and subsequent postmarketing observational study[J]. Arthritis Res Ther, 2014, 16(1): R24. DOI:10.1186/ar4452
[7]
SMOLEN JS, EMERY P, FLEISCHMANN R, et al. Adjustment of therapy in rheumatoid arthritis on the basis of achievement of stable low disease activity with adalimumab plus methotrexate or methotrexate alone:the randomised controlled OPTIMA trial[J]. Lancet, 2014, 383(9914): 321-332. DOI:10.1016/S0140-6736(13)61751-1
[8]
TAKEUCHI T, YAMANAKA H, ISHIGURO N, et al. Adalimumab, a human anti-TNF monoclonal antibody, outcome study for the prevention of joint damage in Japanese patients with early rheumatoid arthritis:the HOPEFUL 1 study[J]. Ann Rheum Dis, 2014, 73(3): 536-543. DOI:10.1136/annrheumdis-2012-202433
[9]
KAVANAUGH A, FLEISCHMANN RM, EMERY P, et al. Clinical, functional and radiographic consequences of achieving stable low disease activity and remission with adalimumab plus methotrexate or methotrexate alone in early rheumatoid arthritis:26-week results from the randomised, controlled OPTIMA study[J]. Ann Rheum Dis, 2013, 72(1): 64-71. DOI:10.1136/annrheumdis-2011-201247
[10]
ENG GP, BOUCHELOUCHE P, BARTELS EM, et al. Anti-drug antibodies, drug levels, interleukin-6 and soluble tnf receptors in rheumatoid arthritis patients during the first 6 months of treatment with adalimumab or infliximab:a descriptive cohort study[J]. PLoS One, 2016, 11(9): e0162316. DOI:10.1371/journal.pone.0162316
[11]
YUE C, YOU X, ZHAO L, et al. The effects of adalimumab and methotrexate treatment on peripheral Th17 cells and IL-17/IL-6 secretion in rheumatoid arthritis patients[J]. Rheumatol Int, 2010, 30(12): 1553-1557. DOI:10.1007/s00296-009-1179-x
[12]
AERTS NE, EBO DG, BRIDTS CH, et al. Flow cytometric analysis of phospho-p38 mitogen-activated kinase(MAPK):p38 MAPK does not mediate the effect of adalimumab on peripheral T cell cytokine production in rheumatoid arthritis[J]. Cytokine, 2009, 47(3): 178-184. DOI:10.1016/j.cyto.2009.06.008