第二军医大学学报  2018, Vol. 39 Issue (2): 182-187   PDF    
免疫复合物抑制B淋巴细胞内Toll样受体9激活的JNK和p38通路
钱莉1, 董改琴2, 吴梦芸1, 荣于馨1, 陈文艳1, 刘阳1, 叶枫1, 刘露1     
1. 扬州大学医学院转化医学研究院, 扬州 225001;
2. 扬州大学附属医院儿科, 扬州 225001
摘要: 目的 考察B淋巴细胞内免疫复合物(IC)对Toll样受体9(TLR9)激动剂CpG寡脱氧核苷酸(ODN)诱导的CD40和CD80高表达信号通路的抑制作用。方法 给小鼠腹腔注射CpG ODN和IC后,用免疫磁珠法分选小鼠脾脏CD19+B淋巴细胞,流式细胞术检测B淋巴细胞表面CD40和CD80的表达。免疫磁珠法分选野生型和免疫球蛋白G Fcγ段受体Ⅱb(FcγRb)缺陷小鼠脾脏B淋巴细胞,体外用CpG ODN和(或)IC刺激后,蛋白质印迹法检测细胞内相关蛋白激酶的磷酸化水平。用JNK抑制剂(SP600125,50 μmol/L)和p38抑制剂(SB203580,20 mg/L)处理后,流式细胞术检测CpG ODN活化B淋巴细胞表面CD40和CD80的表达。结果 体内实验结果显示,IC抑制CpG ODN活化B淋巴细胞表面CD40和CD80的表达(P均 < 0.05)。IC抑制B淋巴细胞内CpG ODN诱导的JNK和p38磷酸化水平,但不能抑制FcγRb缺陷小鼠B淋巴细胞JNK和p38的磷酸化水平。SP600125和SB203580处理后,CpG ODN活化B淋巴细胞表面CD40和CD80的表达均下调(P均 < 0.01)。结论 B淋巴细胞内IC通过抑制JNK和p38通路抑制TLR9激动剂CpG ODN诱导的CD40和CD80表达。
关键词: 免疫复合物     Toll样受体     Fc片段     B淋巴细胞     分化群    
Immune complex inhibits Toll-like receptor 9-activated JNK and p38 pathways in B lymphocytes
QIAN Li1, DONG Gai-qin2, WU Meng-yun1, RONG Yu-xin1, CHEN Wen-yan1, LIU Yang1, YE Feng1, LIU Lu1     
1. Institute of Translational Medcine, Medical College of Yangzhou University, Yangzhou 225001, Jiangsu, China;
2. Department of Pediatrics, Affiliated Hospital of Yangzhou University, Yangzhou 225001, Jiangsu, China
Supported by National Natural Science Foundation of China (81001308, 81373130, 81771689), Natural Science Foundation of Jiangsu Province (BK2010315), and Young Academic Leaders Project of Yangzhou University.
Abstract: Objective To explore the inhibitory effect of immune complex (IC) on the signal pathways of high-expressed CD40 and CD80 induced by Toll-like receptor (TLR9) agonist CpG oligodeoxynucleotide (ODN) in B lymphocytes. Methods The mice were intraperitoneally injected with CpG ODN or IC plus CpG ODN, and the spleen CD19+ B lymphocytes were sorted by magnetic-activated cell sorting (MACS). The expressions of CD40 and CD80 on the B lymphocytes were detected by flow cytometry. The spleen B lymphocytes were isolated from wild type and immunoglobulin G Fcγ receptor Ⅱb (FcγRb) knockout mice by MACS. After the isolated cells were stimulated with CpG ODN or IC plus CpG ODN in vitro, the phosphorylation levels of related protein kinases were detected in the B lymphocytes by Western blotting. Following CpG ODN stimulation, the B lymphocytes were treated with JNK p38 inhibitor SP600125 (50 μmol/L) or p38 inhibitor SB203580 (20 mg/L), and then the CD40 and CD80 expression levels on the CpG ODN-activated B lymphocytes were detected by flow cytometry. Results IC inhibited CD40 and CD80 expressions on the CpG ODN-activated B lymphocytes in vivo (both P < 0.05). IC inhibited the phosphorylation levels of JNK and p38 induced by CpG ODN in B lymphocytes, but did not inhibit them in the B lymphocytes from FcγRb-/- mice. The CD40 and CD80 expressions on the CpG ODN-activated B lymphocytes were significantly decreased after treated with SP600125 and SB203580 (both P < 0.01). Conclusion IC can inhibit the CD40 and CD80 expressions induced by TLR9 agonist CpG ODN through inhibiting the JNK and p38 pathways in B lymphocytes.
Key words: immune complex     Toll-like receptor 9     Fc fragment     B lymphocyte     cluster of differentiation    

Toll样受体9(Toll-like receptor 9,TLR9)可以识别细菌和病毒DNA中的非甲基化CpG基序,在机体清除细菌和病毒的防御机制中发挥重要作用,但过度的TLR9应答会对机体造成损害[1-2]。TLR9与系统性红斑狼疮(system lupus erythmatosis,SLE)等自身免疫病的发生和发展密切相关[3],如SLE患者外周血中B淋巴细胞表达高水平的TLR9且与血清中抗双链DNA抗体水平呈正相关[4];特异性阻断狼疮小鼠模型中TLR9的表达,导致血清中抗双链DNA抗体和抗Sm抗体的水平均降低[5]。因此,负向调节B淋巴细胞上的TLR9信号可能为自身免疫性疾病的治疗提供新思路。

Fcγ段受体(Fcγ receptor,FcγR)Ⅱb是免疫球蛋白(immune globulin,Ig)G的FcγR中唯一的抑制性受体[6]。免疫复合物(immune complex,IC)与B淋巴细胞表面的FcγRb交联可以抑制B淋巴细胞活化及产生抗体[7]。我们前期研究发现,IC还能够通过FcγRb抑制TLR9介导的免疫应答,如体外实验发现IC通过FcγRb抑制B淋巴细胞TLR9配体CpG寡脱氧核苷酸(oligodeoxynucleotide,ODN)刺激的CD40和CD80高表达[8]。本研究进一步探讨IC对TLR9的这一负向调节作用在体内实验中是否同样存在,以及IC通过抑制哪些TLR9信号通路从而抑制CD40和CD80的表达。

1 材料和方法 1.1 材料与试剂

C57BL/6小鼠购自扬州大学实验动物中心;背景品系为C57BL/6的FcγRb缺陷小鼠购自美国杰克逊实验室,由扬州大学医学院转化医学研究院保种[实验动物使用许可证号:SYXK(苏)2012-0029]。CpG ODN由生工生物工程(上海)股份有限公司合成,序列:5' -TCC ATG ACG TTC CTG ACG TT-3' ,序列全部硫代化,经去内毒素纯化后使用;抗小鼠CD19磁珠购自美国美天旎生物技术有限公司;抗CD19、抗CD40和抗CD80的荧光标记抗体均购自美国Biolegend公司;细胞外调节蛋白激酶(extracellular regulated protein kinase,ERK)抑制剂PD98059、c-Jun氨基末端蛋白激酶(c-Jun N-terminal protein kainse,JNK)抑制剂SP600125和p38抑制剂SB203580均购自上海碧云天生物技术有限公司(货号分别为S1805、S1876、S1863);兔抗p-JNK单克隆抗体、兔抗p-ERK单克隆抗体、兔抗p-p38单克隆抗体和兔抗β-actin单克隆抗体均购自美国Cell Signaling公司(货号分别为4671、4370、4511、4970)。

1.2 IC的制备

参照文献[8]方法进行,简述如下:IC由卵清蛋白(ovalbumin,OVA)抗原与抗OVA按1:10的质量比混合,37 ℃孵育1 h后形成。体外刺激B淋巴细胞的IC为10 μg/mL OVA抗原和100 μg/mL抗OVA混合形成;体内实验中每只小鼠的IC为100 μg OVA抗原和1 mg抗OVA混合形成。

1.3 IC和CpG ODN体内注射

小鼠腹腔注射IC 24 h后腹腔注射10 μg CpG ODN,注射CpG ODN 24 h后取小鼠脾脏,用免疫磁珠法分选获得脾脏CD19+ B淋巴细胞[9],并检测其表面CD40和CD80的表达。

1.4 脾脏B淋巴细胞的分选

于无菌条件下取小鼠脾脏,研磨后收集单细胞悬液,用Tris-NH4Cl溶液裂解红细胞,PBS洗涤1次,用抗CD19磁珠分选后,流式细胞术测定CD19+ B淋巴细胞纯度为95%左右。

1.5 流式细胞术检测B淋巴细胞表面CD40和CD80表达

待检细胞中加入异硫氰酸荧光素(fluorescein isothiocyanate,FITC)-抗CD19和藻红蛋白(P-phycoerythrin,PE)-抗CD40或PE-抗CD80,抗体的终浓度均为1 μg/mL,于4 ℃中放置20 min后用PBS洗涤,重悬于300 μL PBS中,上机检测并分析。

1.6 蛋白质印迹法检测蛋白磷酸化

将B淋巴细胞按2×106/mL的密度铺入细胞培养板,用IC和(或)CpG ODN(终浓度0.3 μmol/L)处理30 min,用蛋白裂解液提取细胞总蛋白,聚丙烯酰胺凝胶电泳分离蛋白,后转移至硝酸纤维素膜上,用5%脱脂奶粉溶液室温封闭2 h,加入p-JNK、p-ERK、p-p38和β-actin一抗稀释液(稀释比例均为1:1 000),4 ℃静置孵育过夜,PBST洗涤后加入相应二抗,室温孵育2 h,PBST洗涤后用增强化学发光法显色。

1.7 抑制剂处理细胞

将B淋巴细胞按2×105/孔的密度铺至96孔板,分别加入10 μmol/L PD98059、50 μmol/L SP600125和20 mg/L SB203580,同时设加DMSO作为对照组,处理细胞1 h后,再用0.3 μmol/L CpG ODN刺激细胞24 h,收集细胞用流式细胞术检测其表面CD40和CD80的表达情况。

1.8 统计学处理

采用SPSS 12.0软件对数据进行录入和处理,正态分布且方差齐性的计量资料以x±s表示,组间两两比较采用t检验。检验水准(α)为0.05。

2 结果 2.1 IC抑制CpG ODN活化B淋巴细胞表面CD40和CD80的表达

结果如图 1所示,体内注射CpG ODN能够上调B淋巴细胞表面CD40和CD80的表达(P均<0.05),IC预处理可抑制CpG ODN活化的B淋巴细胞表面CD40和CD80表达上调(P均<0.05)。

图 1 IC体内抑制CpG ODN活化B淋巴细胞表面CD40和CD80表达上调 Fig 1 IC inhibits the up-regulated CD40 and CD80 expressions on CpG ODN-activated B lymphocytes in vivo IC: Immune complex; ODN: Oligodeoxynucleotide; MFI: Median fluorescent intensity. *P < 0.05. n=3, x±s

2.2 IC抑制CpG ODN活化B淋巴细胞JNK和p38的磷酸化

CpG ODN活化B淋巴细胞后不能明显诱导ERK磷酸化,但可以诱导JNK和p38磷酸化。而IC能够抑制CpG ODN活化B淋巴细胞JNK和p38磷酸化(图 2)。

图 2 IC抑制CpG ODN活化B淋巴细胞JNK和p38磷酸化 Fig 2 IC inhibits JNK and p38 phosphorylation in CpG ODN-activated B lymphocytes IC: Immune complex; ODN: Oligodeoxynucleotide

2.3 IC通过FcγRb抑制CpG ODN活化B淋巴细胞JNK和p38磷酸化

结果如图 3所示,IC对FcγRb缺陷小鼠来源的CpG ODN活化B淋巴细胞JNK和p38磷酸化水平无明显抑制作用,提示IC抑制CpG ODN活化B淋巴细胞JNK和p38的磷酸化依赖于FcγRb

图 3 IC对FcγRb缺陷小鼠来源CpG ODN活化B淋巴细胞JNK和p38磷酸化的作用 Fig 3 Effects of IC on JNK and p38 phosphorylation in CpG ODN-activated B lymphocytes from FcγRb-/- mice IC: Immune complex; ODN: Oligodeoxynucleotide; FcγRⅡb: Fcγ receptor Ⅱb

2.4 JNK和p38抑制剂下调CpG ODN活化B淋巴细胞表面CD40和CD80的表达

结果如图 4所示,JNK抑制剂(SP600125)和p38抑制剂(SB203580)均下调CpG ODN活化B淋巴细胞表面CD40和CD80的表达(P均<0.01),提示B淋巴细胞内CpG ODN通过JNK和p38通路调控CD40和CD80的表达。而ERK抑制剂(PD98059)未见相同作用。

图 4 JNK和p38对CpG ODN活化B淋巴细胞表面CD40和CD80表达的影响 Fig 4 Effects of JNK and p38 on CD40 and CD80 expressions in CpG ODN-activated B lymphocytes A: Flow cytometry results; B: Quantitative analysis. PD98059: ERK inhibitor, 10 μmol/L; SP600125: JNK inhibitor, 50 μmol/L; SB203580: p38 inhibitor, 20 mg/L. ODN: Oligodeoxynucleotide; DMSO: Dimethyl sulphoxide; MFI: Median fluorescent intensity. **P < 0.01. n=3, x±s

3 讨论

静脉注射IgG治疗多种自身免疫性疾病已取得良好的疗效[10],IgG与可溶性抗原形成可溶性IC后能模拟静脉注射IgG的效应用于治疗某些自身免疫性疾病[11]。静脉注射Ig能负向调节树突状细胞表面CD80和CD86表达[12],体外用IC可以抑制CpG ODN活化B淋巴细胞表面CD40和CD80表达上调[8]。本研究结果显示,体内注射IC同样能抑制CpG ODN活化B淋巴细胞表面CD40和CD80表达。有文献报道,B淋巴细胞和T淋巴细胞表面CD40与CD40配体(CD40L)相互作用对SLE自身抗体的产生有重要作用[13],抗CD40L能阻断SLE小鼠自身抗体的产生[14],提示阻断CD40-CD40L通路可能对治疗SLE有重要作用。

所有B淋巴细胞亚群均表达TLR9且能被TLR9配体活化[15]。TLR9不仅能被病毒和细菌DNA非甲基化CpG基序活化,同样也能被人工合成短的单链ODN活化[16]。因而,无论来源如何,所有富含CpG基序均能活化B淋巴细胞。B淋巴细胞TLR9介导的信号通路与树突状细胞类似,即TLR9配体作用于TLR9后引起丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)和NF-κB途径活化[17]。本实验发现,CpG ODN活化B淋巴细胞后引起NF-κB途径(数据未显示)以及JNK和p38-MAPK途径的活化,但不能明显诱导ERK-MAPK途径的活化;IC作用后CpG ODN诱导的NF-κB活化并没有受到明显影响(数据未显示),而JNK和p38的活化被明显抑制,提示IC并不是通过抑制NF-κB通路抑制CD40和CD80的表达,而有可能通过抑制JNK和p38-MAPK途径从而对CD40和CD80的表达发挥抑制作用。本实验结果表明,JNK和p38抑制剂能下调CpG ODN活化B淋巴细胞表面CD40和CD80的表达,提示JNK和p38-MAPK途径确实参与了对CD40和CD80表达的调控。

IC抑制TLR9通路的机制目前还不清楚。前期检测了IC刺激后的B淋巴细胞内某些蛋白激酶的磷酸化水平,发现IC刺激不引起脾酪氨酸激酶和Bruton酪氨酸激酶的活化,但能使酪氨酸激酶Lyn活化。抑制Lyn会导致TLR4介导的CD40表达进一步增加,提示IC通过活化Lyn抑制TLR4介导的CD40高表达[18]。有文献报道,Lyn可以磷酸化接头蛋白Dok,维持含SH2区域肌醇5'磷酸酶1的活化,从而抑制NF-κB通路的活化[19-20]。此外,前期研究还发现激活B淋巴细胞Lyn可以抑制NF-κB通路的活化[21]。那么IC能否通过活化Lyn抑制TLR9通路中的JNK和p38-MAPK信号通路,从而抑制CD40和CD80的表达,仍需要进一步的实验研究。

综上所述,本研究发现IC与B淋巴细胞的FcγRⅡb结合抑制TLR9激动剂CpG ODN诱导的JNK和p38-MAPK途径,从而抑制B淋巴细胞表面CD40和CD80的表达。本实验结果有助于更全面地认识IC对TLR介导的免疫应答负向调节机制,也为治疗B淋巴细胞内过度TLR9应答提供了线索。

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