重组人Ⅱ型胶原250-270多肽的鉴定及其特异性免疫调节作用的研究
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摘要
类风湿关节炎(rheumatoid arthritis,RA)是一种以多关节侵蚀破坏为特征的全身性炎性自身免疫病。发病率约为0.3～1.0%,致残率和死亡率均高。RA的病因及发病机制目前尚不完全清楚,迄今尚无根治疗法。
口服耐受是指口服某种抗原物质后机体对该抗原再次接触时表现的一种免疫无应答状态,可以作为治疗许多疾病特别是自身免疫性疾病的手段,并具有无毒、方便和抗原特异性的特点,因此,口服耐受成为免疫学和临床研究的热点。胶原诱导性关节炎(collagen-induced arthritis,CIA)是采用Ⅱ型胶原(typeⅡcollagen,CⅡ)免疫胶原敏感品系的鼠类,从而诱导多关节炎症的发生。CIA动物模型的症状和关节病理变化与RA相似,被广泛用于研究人类RA发病机制和探索治疗新方法。已有研究表明,口服CⅡ或CⅡ免疫显性多肽可抑制CIA的发展。合成CⅡ250-270多肽(synthesis collagen typeⅡpolypeptide 250-270,syCⅡ250-270)内含有免疫显性T细胞表位CⅡ260-270,可诱导敏感品系小鼠发生CIA和免疫耐受,对研究CIA和RA的发病机理和治疗都具有重要意义。
化学合成多肽技术复杂、代价昂贵、产量少和难于标准化,无法满足基础研究和临床应用的需要,而采用基因工程技术生产的重组多肽产品则可以解决这一问题。因此,本研究对重组人CⅡ250-270基因进行表达,获得了重组人CⅡ250-270多肽(recombinant human collagen typeⅡpeptide 250-270,rh CⅡ250-270),对此肽段进行分离、纯化和鉴定。
虽然,应用口服抗原诱导免疫耐受治疗自身免疫性疾病,在许多动物模型中己取得了令人满意的结果。但在临床以此方法治疗RA患者时,结果却不尽人意。为探讨口服耐受在治疗RA中确切机制和效果,本研究通过给予CIA小鼠早期口服低剂量CⅡ250-270多肽,观察其对CIA的特异性细胞和体液免疫反应的调节作用。同时,建立了在单细胞水平上对抗原特异性淋巴细胞的检测体系,使用流式细胞仪同时分析溴代脱氧尿嘧啶核苷(bromodeoxyuridine,BrdU)掺入、细胞内细胞因子、细胞表面活化标志并采用酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测特异性抗体和酶联免疫斑点测定法(enzyme-linked immunospot assay,ELISPOT)检测抗原特异性抗体形成细胞的综合检测方法,有助于更好地理解口服耐受的免疫学机制。
目的
获得大量rhCⅡ250-270多肽,鉴定并确认rhCⅡ250-270多肽与预期的相符。建立一种评估抗原特异性细胞和体液免疫反应的免疫标记和方法,有助于口服耐受在疾病治疗的研究。研究CⅡ250-270多肽在RA和CIA发病中作用及其机制,并为应用口服CⅡ250-270多肽治疗RA提供理论依据。
方法
1. rhCⅡ250-270多肽的表达、纯化和鉴定:在E.coli BL21中表达rhCⅡ250-270蛋白多肽,用亲和层析法分离纯化。用限制性酶切法、聚合酶链式反应(polymerase chain reaction,PCR)法、基因序列测定法、琼脂糖凝胶电泳法、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法(sodium dodecyl sulphate-polyacrylamine gel electrophoresis,SDS-PAGE)和免疫印迹法(Western Blotting)等方法在基因和蛋白水平上对rhCⅡ250-270多肽进行鉴定。
2. rhCⅡ250-270多肽免疫原性的研究:以不同浓度(0.5、1.0、2.0mg/mL)的6聚rh CⅡ250-270和(20、200、2000μg/mL)CⅡ刺激体外培养的RA患者PBMC,等浓度的PBS作为阴性对照,PHA刺激组作为阳性对照。流式细胞仪测定刺激前后各组培养细胞中T细胞表面活化标志性抗原(CD69,CD25)的表达及5-溴脱氧尿嘧啶(BrdU)在DNA中的掺入;rhCⅡ250-270免疫CIA敏感小鼠DBA/1,比较与CⅡ和合成CⅡ250-270免疫后在体内血清特异性抗体水平的差异。
3.建立CIA模型和诱导口服耐受:鸡Ⅱ型胶原(chicken collagenⅡ,CCⅡ)在DBA/1小鼠(H-2q单倍型)尾部皮内注射,定期观察并进行关节评分;在CⅡ免疫小鼠前一周,给予口服rhCⅡ250-270多肽(0.5mg/mL)或合成多肽(0.1mg/mL),分别两次。
4. rhCⅡ250-270多肽对实验动物脾脏特异性T淋巴细胞活化及增殖实验:取加强免疫后7d治疗组和CIA对照组的小鼠脾脏淋巴细胞,体外经CⅡ250-270、CⅡ和PHA刺激培养后,采用BrdU掺入法和流式细胞术同时测定CⅡ特异性T细胞增殖情况、细胞膜表面标志;并通过流式细胞术测定外周血中CD4+T细胞的细胞内细胞因子干扰素-γ(interferon-γ,IFN-γ)、白细胞介素-4 (interleukin-4,IL-4)的水平分析辅助性T细胞1(helper T cell 1,Th1)和Th2亚群的变化。
5. rhCⅡ250-270多肽对实验动物脾脏特异性B淋巴细胞反应:用ELISA法测定加强免疫后7d各组小鼠血清中抗CⅡ抗体的表达;用ELISPOT法检测加强免疫后7d各组小鼠脾脏淋巴细胞特异性抗CⅡ抗体形成脾细胞。
6.组织病理学检查:加强免疫后6周,取小鼠的炎症关节进行HE染色。观察关节病变的变化情况并对其进行评分。
结果
1.纯化的rhCⅡ250-270多肽,大小约43 kD,纯度为89.3%。EcoRⅠ和SalⅠ双酶切法和PCR法得到的DNA大小约为500bp和650bp,与预期的522bp和679bp基本相符,基因序列测定证实基因DNA序列与设计的完全相符。SDS-PAGE结果显示rhCⅡ250-270多肽目的蛋白条带的大小约为43 kD,与设计预期相符。Western Blotting结果显示GST融合蛋白上存在CⅡ片段。
2. rhCⅡ250-270多肽能刺激RA患者PBMC中的特异性淋巴细胞活化和增殖反应,同样可以诱导CIA敏感小鼠特异性抗体的活化,血清抗体水平与CⅡ和合成CⅡ250-270免疫组无显著性差异。提示重组CⅡ250-270具有CⅡ及合成CⅡ250-270相似的免疫学活性。
3. DBA/1小鼠口服重组CⅡ250-270或合成CⅡ250-270多肽后, CIA的发病率低于对照组(口服PBS),关节炎评分也较对照组明显降低,关节炎发病延迟,且体重未减轻,而CIA对照组小鼠的体重则有所下降。表明CⅡ250-270多肽可在一定程度上减轻CIA小鼠病情。
4.口服CⅡ250-270多肽可诱导对小鼠脾细胞中的抗原特异性T细胞的活化增殖及IFN-γ的分泌的抑制。口服CⅡ250-270多肽小鼠脾细胞中CD3+CD25+抗原特异性T细胞的百分比(18.76%)比对照组(30.67%)明显下降,P<0.01;口服rhCⅡ250-270多肽0.5mg组和syCⅡ250-270多肽0.1mg组CD3+BrdU+ T细胞的百分比分别为27.67%和24.79%,明显低于对照组(50.46%),P<0.01;口服CⅡ250-270多肽组脾细胞用rhCⅡ250-270多肽再刺激后,分泌IFN-γ的阳性细胞率为9.86%和12.89%,明显低于对照组的24.36%,P<0.01。
5.口服CⅡ250-270小鼠血清中抗CⅡ和抗CⅡ250-270的IgG抗体水平[A值分别为(0.82±0.02)、(0.84±0.04)]比CⅡ免疫对照组[抗CⅡ的IgG抗体水平,A值为(1.01±0.06)]显著降低,而且特异性抗CⅡ250-270的IgG抗体反应在口服多肽组被明显抑制(P<0.01)。同时,口服CⅡ250-270小鼠的CⅡ和CⅡ250-270特异性抗体形成脾细胞的增殖频率分别为(158±9计数/孔)、(181±10计数/孔),比CⅡ免疫对照组[CⅡ特异性抗体形成脾细胞的增殖频率为(247±16计数/孔)]也显著减少(P<0.05),
6.组织病理学观察,口服CⅡ250-270多肽,CIA小鼠关节破坏情况也得到了改善。炎细胞浸润、滑膜增生和血管翳的形成,软骨及骨组织损伤,口服rhCⅡ250-270多肽0.5mg组和syCⅡ250-270多肽0.1mg组,比口服PBS对照组,组织病理学评分均有所降低。
结论
本研究对rhCⅡ250-270多肽进行了较全面的鉴定,发现此肽段具有较强的免疫原性,可刺激RA患者PBMC中的特异性淋巴细胞活化和增殖反应以及诱导CIA敏感小鼠特异性抗体的活化,并且和合成CⅡ250-270多肽在抑制CIA小鼠的特异性细胞免疫和体液免疫上具有同样的效果。本实验还建立了在单细胞水平上同时检测分析抗原特异性细胞免疫和体液免疫的实验体系。这些研究将有助于阐明CIA的口服耐受机制,为应用口服CⅡ250-270多肽这一新的RA治疗方法提供理论依据。
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by a chronic inflammation of the synovial membrane which is associated with destruction of cartilage and bone. It is a common human autoimmune disease with a prevalence of about 0.3～1.0%. The pathogenesis is not completely understood.
‘Oral tolerance’has classically been defined as the specific suppression of cellular and/or humoral immune responses to an antigen by prior administration of the antigen by the oral route. Mucosal tolerance is an attractive approach for treatment of autoimmune and inflammatory diseases because of lack of toxicity, ease of administration over time, and antigen-specific mechanisms of action. Oral tolerance induced by typeⅡcollagen (CⅡ) and synthesis polypeptide 250-270 (syCⅡ250-270) peptide have been demonstrated to be a nondangerous, effective way for preventing harmful inflammatory in animal model of RA. However, it is more difficult to extract CⅡfrom animal cartilage tissue. Furthermore, the source of material is much limited to supply the demand of basal and clinical application for research and treatment of RA and the other diseases. The objective of this paper is to high-performance express the recombinant polymerized gene encoding human collagen typeⅡpolypeptide 250-270 (rhCⅡ250-270). Then, in order to proved that rhCⅡ2 50-270 characterize with predicted immunological characterization, we had explored its ability to stimulate the T cells of PBMC in patients with RA and detected autoantibodies to rhCⅡ250-270 in the sera of rhCⅡ250-270-immunized mice.
It has been demonstrated that oral antigen administration can suppress animal models of autoimmune diseases. Unfortunately, although positive results have been observed in phase II trials, no effect was observed in phase III trials of CⅡin rheumatoid arthritis. In order to further investigat the modulation of rhCⅡ250-270 peptide, with immunodominant epitopes on special cellular and humoral immune response in the course of oral administration by in vitro and in vivo experiment, we induced low doses of orally administered rhCⅡ250-270 in early disease in mice CIA. The cellular events that characterize lymphocyte responses to specific stimuli include expression of cell surface activation antigens (CD69 or CD25), cytokine production (IFN-γ, IL-4) and proliferation (BrdU incorporation) were simultaneous detected. The antigen-specific antibody and antibody-forming cells were detected by ELISA and ELISPOT, respectively.
Methods
1. The human immunodominant peptide of CⅡ250-270 was expressed in E.coli.and purified by affinity chromatography for the first time. Fusion protein was expressed in pGEX-4T-1 and isolated and purified by Glutathione Sepharose? affinity chromatography.
2. rhCⅡ2 50-270 was proved to characterize with predicted immunological characterization by flow cytometry analysis of activation and proliferation of antigen specific T cells including cellsurface markers (e.g. CD3, CD69 and CD25) and BrdU incorporation, and by the activity of IgG rhCⅡ2 50-270-specific antibody in sera in mice immunized with rhCⅡ2 50-270.
3. DBA/1 mice were immunized with emulsified chicken CⅡor rhCⅡ2 50-270 in complete Freund’s adjuvant by intradermal injection to induce the mice model of arthritis; Oral tolerance was induced by oral rhCⅡ2 50-270 or syCⅡ2 50-270 in different doses (5 mg/mL, 1 mg/mL, respectively, twice before the induction phase of CIA).
4. The lymphocytes were obtained from spleen of mice killed at 7 days of booster immunization, and the antigen specific reactive T cells were stimulated by CⅡor CⅡ250-270 in vitro. The proliferation response and phenotype were analyzed by BrdU incorporation and fluorescence-activated cell sorter (FACS). Intracellular cytokines (IFN-γ, IL-4) and surface antigen (CD4, CD3, CD69 and CD25 )of splenocytes were assayed by FACS.
5. The frequency of IgG anti-CⅡ, anti-rhCⅡ250-270, and anti-syCⅡ( 250-270 antibody-forming spleen cells was measured with ELISPOT; ELISA was used to determine antigen-specific antibodies in sera in mice.
6. To observe the effects of oral CⅡ250-270 on joint structure at the histologic level, inflamed joints were stained with hematoxylin and eosin and evaluated after mice was sacrificed at 3 weeks after booster immunization.
Results
1. rhCⅡ2 50-270 had been successfully expressed and purified. The relative molecular mass(Mr)of expressed product was 43 KD which is in accord with predicted. The purity of purified fusion protein reached 89.3 percent assessed by SDS-PAGE gel thin-layer scanning.
2. rhCⅡ( 250-270 in vitro could stimulate the response of specific lymphocyte in RA patients PBMC including the increase of surface activation antigen marker CD69, CD25 expression and DNA synthesis. rhCⅡ2 50-270 in vivo could induced antigen-specific antibody in DBA/1 mice. As well as native CⅡ, rhCⅡ( 250-270 could be recognized as immunogenic antigens by T and B cells.
3. We found that CⅡ(250-270 to some extent reduced the severity of CIA through the utilization of immunological regulatory mechanisms induced by feeding rhCⅡ250-270 to CIA. The general condition of the mice was improved.
4. Splenocytes from mice fed rhCⅡ250-270 and syCⅡ250-270 stimulated significantly less specific splenocyte activation compare with CIA group. The percentages of CD3+CD25+ cell from mice fed CⅡ250-270 were reduced by at least 18.76% stimulated by rhCⅡ250-270. The percentages of CD3+BrdU+ cell from mice fed CⅡ250-270 after stimulation with antigens in vitro significantly less than CIA group. Those from mice fed rhCⅡ250-270 (5mg/mL),and syCⅡ250-270 (1mg/mL) were 27.67%, 24.79% and stimulated by rhCⅡ250-270, respectively, while in CIA was up to 50.46%. Splenocytes from the fed polypeptide groups of mice that were secreted significantly less IFN-γ, which were 9.86% and 12.89% stimulated by rhCⅡ250-270, respectively, than splenocytes from CIA (24.36%).
5. The level of CⅡ- and CⅡ2 50-270 -specific IgG in serum from mice fed with rhCⅡ250-270 were (0.82±0.02) and (0.84±0.04) respectively,and significantly lower than those of collagen-induced arthritis (CIA) control group. The anti-CⅡ250-270 antibody responses were obviously suppressed (P<0.01).The frequency of antibody-forming cells in the spleen from rhCⅡ250-270-fed mice were (158±9 counts/well ) and (181±10 counts/well)respectively, and also significantly were reduced when compared with that in CIA control group (247±16 counts/well ).
6. Consistent with the lack of treatment effect, oral rhCⅡ250-270 in doses of 5mg/mL have lower histologic scores for synovial inflammation or pannus invasion, degrees of cartilage damage and bone damage at either the joints.
Generally, the human immunodominant peptide of CⅡ250-270 was expressed and purified for the first time in this paper. Then, it was proved to characterize with predicted immunological characterization. Further, present study identification that oral rhCⅡ250-270 could induce specific suppress of cellular and humoral in CIA. These findings and the experiment system, together with a better understanding of the mechanisms of oral tolerance and regulation cellular and humoral immune response in CIA, will help the development of innovative therapeutic intervention for RA.
口服耐受是指口服某种抗原物质后机体对该抗原再次接触时表现的一种免疫无应答状态,可以作为治疗许多疾病特别是自身免疫性疾病的手段,并具有无毒、方便和抗原特异性的特点,因此,口服耐受成为免疫学和临床研究的热点。胶原诱导性关节炎(collagen-induced arthritis,CIA)是采用Ⅱ型胶原(typeⅡcollagen,CⅡ)免疫胶原敏感品系的鼠类,从而诱导多关节炎症的发生。CIA动物模型的症状和关节病理变化与RA相似,被广泛用于研究人类RA发病机制和探索治疗新方法。已有研究表明,口服CⅡ或CⅡ免疫显性多肽可抑制CIA的发展。合成CⅡ250-270多肽(synthesis collagen typeⅡpolypeptide 250-270,syCⅡ250-270)内含有免疫显性T细胞表位CⅡ260-270,可诱导敏感品系小鼠发生CIA和免疫耐受,对研究CIA和RA的发病机理和治疗都具有重要意义。
化学合成多肽技术复杂、代价昂贵、产量少和难于标准化,无法满足基础研究和临床应用的需要,而采用基因工程技术生产的重组多肽产品则可以解决这一问题。因此,本研究对重组人CⅡ250-270基因进行表达,获得了重组人CⅡ250-270多肽(recombinant human collagen typeⅡpeptide 250-270,rh CⅡ250-270),对此肽段进行分离、纯化和鉴定。
虽然,应用口服抗原诱导免疫耐受治疗自身免疫性疾病,在许多动物模型中己取得了令人满意的结果。但在临床以此方法治疗RA患者时,结果却不尽人意。为探讨口服耐受在治疗RA中确切机制和效果,本研究通过给予CIA小鼠早期口服低剂量CⅡ250-270多肽,观察其对CIA的特异性细胞和体液免疫反应的调节作用。同时,建立了在单细胞水平上对抗原特异性淋巴细胞的检测体系,使用流式细胞仪同时分析溴代脱氧尿嘧啶核苷(bromodeoxyuridine,BrdU)掺入、细胞内细胞因子、细胞表面活化标志并采用酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测特异性抗体和酶联免疫斑点测定法(enzyme-linked immunospot assay,ELISPOT)检测抗原特异性抗体形成细胞的综合检测方法,有助于更好地理解口服耐受的免疫学机制。
目的
获得大量rhCⅡ250-270多肽,鉴定并确认rhCⅡ250-270多肽与预期的相符。建立一种评估抗原特异性细胞和体液免疫反应的免疫标记和方法,有助于口服耐受在疾病治疗的研究。研究CⅡ250-270多肽在RA和CIA发病中作用及其机制,并为应用口服CⅡ250-270多肽治疗RA提供理论依据。
方法
1. rhCⅡ250-270多肽的表达、纯化和鉴定:在E.coli BL21中表达rhCⅡ250-270蛋白多肽,用亲和层析法分离纯化。用限制性酶切法、聚合酶链式反应(polymerase chain reaction,PCR)法、基因序列测定法、琼脂糖凝胶电泳法、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法(sodium dodecyl sulphate-polyacrylamine gel electrophoresis,SDS-PAGE)和免疫印迹法(Western Blotting)等方法在基因和蛋白水平上对rhCⅡ250-270多肽进行鉴定。
2. rhCⅡ250-270多肽免疫原性的研究:以不同浓度(0.5、1.0、2.0mg/mL)的6聚rh CⅡ250-270和(20、200、2000μg/mL)CⅡ刺激体外培养的RA患者PBMC,等浓度的PBS作为阴性对照,PHA刺激组作为阳性对照。流式细胞仪测定刺激前后各组培养细胞中T细胞表面活化标志性抗原(CD69,CD25)的表达及5-溴脱氧尿嘧啶(BrdU)在DNA中的掺入;rhCⅡ250-270免疫CIA敏感小鼠DBA/1,比较与CⅡ和合成CⅡ250-270免疫后在体内血清特异性抗体水平的差异。
3.建立CIA模型和诱导口服耐受:鸡Ⅱ型胶原(chicken collagenⅡ,CCⅡ)在DBA/1小鼠(H-2q单倍型)尾部皮内注射,定期观察并进行关节评分;在CⅡ免疫小鼠前一周,给予口服rhCⅡ250-270多肽(0.5mg/mL)或合成多肽(0.1mg/mL),分别两次。
4. rhCⅡ250-270多肽对实验动物脾脏特异性T淋巴细胞活化及增殖实验:取加强免疫后7d治疗组和CIA对照组的小鼠脾脏淋巴细胞,体外经CⅡ250-270、CⅡ和PHA刺激培养后,采用BrdU掺入法和流式细胞术同时测定CⅡ特异性T细胞增殖情况、细胞膜表面标志;并通过流式细胞术测定外周血中CD4+T细胞的细胞内细胞因子干扰素-γ(interferon-γ,IFN-γ)、白细胞介素-4 (interleukin-4,IL-4)的水平分析辅助性T细胞1(helper T cell 1,Th1)和Th2亚群的变化。
5. rhCⅡ250-270多肽对实验动物脾脏特异性B淋巴细胞反应:用ELISA法测定加强免疫后7d各组小鼠血清中抗CⅡ抗体的表达;用ELISPOT法检测加强免疫后7d各组小鼠脾脏淋巴细胞特异性抗CⅡ抗体形成脾细胞。
6.组织病理学检查:加强免疫后6周,取小鼠的炎症关节进行HE染色。观察关节病变的变化情况并对其进行评分。
结果
1.纯化的rhCⅡ250-270多肽,大小约43 kD,纯度为89.3%。EcoRⅠ和SalⅠ双酶切法和PCR法得到的DNA大小约为500bp和650bp,与预期的522bp和679bp基本相符,基因序列测定证实基因DNA序列与设计的完全相符。SDS-PAGE结果显示rhCⅡ250-270多肽目的蛋白条带的大小约为43 kD,与设计预期相符。Western Blotting结果显示GST融合蛋白上存在CⅡ片段。
2. rhCⅡ250-270多肽能刺激RA患者PBMC中的特异性淋巴细胞活化和增殖反应,同样可以诱导CIA敏感小鼠特异性抗体的活化,血清抗体水平与CⅡ和合成CⅡ250-270免疫组无显著性差异。提示重组CⅡ250-270具有CⅡ及合成CⅡ250-270相似的免疫学活性。
3. DBA/1小鼠口服重组CⅡ250-270或合成CⅡ250-270多肽后, CIA的发病率低于对照组(口服PBS),关节炎评分也较对照组明显降低,关节炎发病延迟,且体重未减轻,而CIA对照组小鼠的体重则有所下降。表明CⅡ250-270多肽可在一定程度上减轻CIA小鼠病情。
4.口服CⅡ250-270多肽可诱导对小鼠脾细胞中的抗原特异性T细胞的活化增殖及IFN-γ的分泌的抑制。口服CⅡ250-270多肽小鼠脾细胞中CD3+CD25+抗原特异性T细胞的百分比(18.76%)比对照组(30.67%)明显下降,P<0.01;口服rhCⅡ250-270多肽0.5mg组和syCⅡ250-270多肽0.1mg组CD3+BrdU+ T细胞的百分比分别为27.67%和24.79%,明显低于对照组(50.46%),P<0.01;口服CⅡ250-270多肽组脾细胞用rhCⅡ250-270多肽再刺激后,分泌IFN-γ的阳性细胞率为9.86%和12.89%,明显低于对照组的24.36%,P<0.01。
5.口服CⅡ250-270小鼠血清中抗CⅡ和抗CⅡ250-270的IgG抗体水平[A值分别为(0.82±0.02)、(0.84±0.04)]比CⅡ免疫对照组[抗CⅡ的IgG抗体水平,A值为(1.01±0.06)]显著降低,而且特异性抗CⅡ250-270的IgG抗体反应在口服多肽组被明显抑制(P<0.01)。同时,口服CⅡ250-270小鼠的CⅡ和CⅡ250-270特异性抗体形成脾细胞的增殖频率分别为(158±9计数/孔)、(181±10计数/孔),比CⅡ免疫对照组[CⅡ特异性抗体形成脾细胞的增殖频率为(247±16计数/孔)]也显著减少(P<0.05),
6.组织病理学观察,口服CⅡ250-270多肽,CIA小鼠关节破坏情况也得到了改善。炎细胞浸润、滑膜增生和血管翳的形成,软骨及骨组织损伤,口服rhCⅡ250-270多肽0.5mg组和syCⅡ250-270多肽0.1mg组,比口服PBS对照组,组织病理学评分均有所降低。
结论
本研究对rhCⅡ250-270多肽进行了较全面的鉴定,发现此肽段具有较强的免疫原性,可刺激RA患者PBMC中的特异性淋巴细胞活化和增殖反应以及诱导CIA敏感小鼠特异性抗体的活化,并且和合成CⅡ250-270多肽在抑制CIA小鼠的特异性细胞免疫和体液免疫上具有同样的效果。本实验还建立了在单细胞水平上同时检测分析抗原特异性细胞免疫和体液免疫的实验体系。这些研究将有助于阐明CIA的口服耐受机制,为应用口服CⅡ250-270多肽这一新的RA治疗方法提供理论依据。
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by a chronic inflammation of the synovial membrane which is associated with destruction of cartilage and bone. It is a common human autoimmune disease with a prevalence of about 0.3～1.0%. The pathogenesis is not completely understood.
‘Oral tolerance’has classically been defined as the specific suppression of cellular and/or humoral immune responses to an antigen by prior administration of the antigen by the oral route. Mucosal tolerance is an attractive approach for treatment of autoimmune and inflammatory diseases because of lack of toxicity, ease of administration over time, and antigen-specific mechanisms of action. Oral tolerance induced by typeⅡcollagen (CⅡ) and synthesis polypeptide 250-270 (syCⅡ250-270) peptide have been demonstrated to be a nondangerous, effective way for preventing harmful inflammatory in animal model of RA. However, it is more difficult to extract CⅡfrom animal cartilage tissue. Furthermore, the source of material is much limited to supply the demand of basal and clinical application for research and treatment of RA and the other diseases. The objective of this paper is to high-performance express the recombinant polymerized gene encoding human collagen typeⅡpolypeptide 250-270 (rhCⅡ250-270). Then, in order to proved that rhCⅡ2 50-270 characterize with predicted immunological characterization, we had explored its ability to stimulate the T cells of PBMC in patients with RA and detected autoantibodies to rhCⅡ250-270 in the sera of rhCⅡ250-270-immunized mice.
It has been demonstrated that oral antigen administration can suppress animal models of autoimmune diseases. Unfortunately, although positive results have been observed in phase II trials, no effect was observed in phase III trials of CⅡin rheumatoid arthritis. In order to further investigat the modulation of rhCⅡ250-270 peptide, with immunodominant epitopes on special cellular and humoral immune response in the course of oral administration by in vitro and in vivo experiment, we induced low doses of orally administered rhCⅡ250-270 in early disease in mice CIA. The cellular events that characterize lymphocyte responses to specific stimuli include expression of cell surface activation antigens (CD69 or CD25), cytokine production (IFN-γ, IL-4) and proliferation (BrdU incorporation) were simultaneous detected. The antigen-specific antibody and antibody-forming cells were detected by ELISA and ELISPOT, respectively.
Methods
1. The human immunodominant peptide of CⅡ250-270 was expressed in E.coli.and purified by affinity chromatography for the first time. Fusion protein was expressed in pGEX-4T-1 and isolated and purified by Glutathione Sepharose? affinity chromatography.
2. rhCⅡ2 50-270 was proved to characterize with predicted immunological characterization by flow cytometry analysis of activation and proliferation of antigen specific T cells including cellsurface markers (e.g. CD3, CD69 and CD25) and BrdU incorporation, and by the activity of IgG rhCⅡ2 50-270-specific antibody in sera in mice immunized with rhCⅡ2 50-270.
3. DBA/1 mice were immunized with emulsified chicken CⅡor rhCⅡ2 50-270 in complete Freund’s adjuvant by intradermal injection to induce the mice model of arthritis; Oral tolerance was induced by oral rhCⅡ2 50-270 or syCⅡ2 50-270 in different doses (5 mg/mL, 1 mg/mL, respectively, twice before the induction phase of CIA).
4. The lymphocytes were obtained from spleen of mice killed at 7 days of booster immunization, and the antigen specific reactive T cells were stimulated by CⅡor CⅡ250-270 in vitro. The proliferation response and phenotype were analyzed by BrdU incorporation and fluorescence-activated cell sorter (FACS). Intracellular cytokines (IFN-γ, IL-4) and surface antigen (CD4, CD3, CD69 and CD25 )of splenocytes were assayed by FACS.
5. The frequency of IgG anti-CⅡ, anti-rhCⅡ250-270, and anti-syCⅡ( 250-270 antibody-forming spleen cells was measured with ELISPOT; ELISA was used to determine antigen-specific antibodies in sera in mice.
6. To observe the effects of oral CⅡ250-270 on joint structure at the histologic level, inflamed joints were stained with hematoxylin and eosin and evaluated after mice was sacrificed at 3 weeks after booster immunization.
Results
1. rhCⅡ2 50-270 had been successfully expressed and purified. The relative molecular mass(Mr)of expressed product was 43 KD which is in accord with predicted. The purity of purified fusion protein reached 89.3 percent assessed by SDS-PAGE gel thin-layer scanning.
2. rhCⅡ( 250-270 in vitro could stimulate the response of specific lymphocyte in RA patients PBMC including the increase of surface activation antigen marker CD69, CD25 expression and DNA synthesis. rhCⅡ2 50-270 in vivo could induced antigen-specific antibody in DBA/1 mice. As well as native CⅡ, rhCⅡ( 250-270 could be recognized as immunogenic antigens by T and B cells.
3. We found that CⅡ(250-270 to some extent reduced the severity of CIA through the utilization of immunological regulatory mechanisms induced by feeding rhCⅡ250-270 to CIA. The general condition of the mice was improved.
4. Splenocytes from mice fed rhCⅡ250-270 and syCⅡ250-270 stimulated significantly less specific splenocyte activation compare with CIA group. The percentages of CD3+CD25+ cell from mice fed CⅡ250-270 were reduced by at least 18.76% stimulated by rhCⅡ250-270. The percentages of CD3+BrdU+ cell from mice fed CⅡ250-270 after stimulation with antigens in vitro significantly less than CIA group. Those from mice fed rhCⅡ250-270 (5mg/mL),and syCⅡ250-270 (1mg/mL) were 27.67%, 24.79% and stimulated by rhCⅡ250-270, respectively, while in CIA was up to 50.46%. Splenocytes from the fed polypeptide groups of mice that were secreted significantly less IFN-γ, which were 9.86% and 12.89% stimulated by rhCⅡ250-270, respectively, than splenocytes from CIA (24.36%).
5. The level of CⅡ- and CⅡ2 50-270 -specific IgG in serum from mice fed with rhCⅡ250-270 were (0.82±0.02) and (0.84±0.04) respectively,and significantly lower than those of collagen-induced arthritis (CIA) control group. The anti-CⅡ250-270 antibody responses were obviously suppressed (P<0.01).The frequency of antibody-forming cells in the spleen from rhCⅡ250-270-fed mice were (158±9 counts/well ) and (181±10 counts/well)respectively, and also significantly were reduced when compared with that in CIA control group (247±16 counts/well ).
6. Consistent with the lack of treatment effect, oral rhCⅡ250-270 in doses of 5mg/mL have lower histologic scores for synovial inflammation or pannus invasion, degrees of cartilage damage and bone damage at either the joints.
Generally, the human immunodominant peptide of CⅡ250-270 was expressed and purified for the first time in this paper. Then, it was proved to characterize with predicted immunological characterization. Further, present study identification that oral rhCⅡ250-270 could induce specific suppress of cellular and humoral in CIA. These findings and the experiment system, together with a better understanding of the mechanisms of oral tolerance and regulation cellular and humoral immune response in CIA, will help the development of innovative therapeutic intervention for RA.
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