南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (08): 1081-1089.doi: 10.12122/j.issn.1673-4254.2020.08.03

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高强度炎症反应诱导关节软骨特异性瓜氨酸化抗原表达

覃桂城，林晓吟，梁培彬，李艳鹏，周 春，Kutty Selva Nandakumar，Rikard Holmdahl   

  • 出版日期:2020-08-20 发布日期:2020-08-20
  • 基金资助:

Strong inflammation is essential for expression of articular cartilage-specific citrullinated antigens

  

  • Online:2020-08-20 Published:2020-08-20

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摘要： 目的 探讨关节软骨蛋白瓜氨酸表位的表达情况，以及瓜氨酸化表位对抗瓜氨酸蛋白抗体（ACPAs）的免疫应答的影响。方法 体外诱导实验：实验组加不同浓度梯度的脂多糖、热灭活细菌（大肠杆菌、金色葡萄球菌）或二型胶原（CII）特异性单克隆抗体等不同刺激物诱导体外培养关节软骨蛋白瓜氨酸化，等量PBS作为对照组，利用特异性结合瓜氨酸化表位的单克隆抗体ACC4（IgG1）进行免疫组织化学染色，ACC1（IgG2a）作为阳性对照，Hy2.15（IgG1）和L243（IgG2a）分别作为同型阴性对照，显微镜下观察瓜氨酸化蛋白表达；体内诱导实验：SD大鼠右膝关节内注射不同浓度脂多糖作为实验组，左膝关节内注射等量PBS为对照组，3 d后取膝关节组织，制成冰冻切片，利用上述抗体进行免疫组化染色，显微镜下观察瓜氨酸化蛋白表达；建立不同种系的胶原诱导性关节炎（CIA）小鼠模型，评价其疾病发生率和严重程度，同时收集小鼠血清与高滴度抗瓜氨酸化抗体的类风湿性关节炎患者血清，采用液相芯片技术检测小鼠及类风湿患者血清中抗瓜氨酸化蛋白抗体水平，并用免疫组织化学检测关节炎小鼠关节软骨瓜氨酸化蛋白的表达水平。结果 特异性结合瓜氨酸化CII 表位的ACC4抗体未能结合至经不同诱导的关节软骨组织；与健康对照组相比，ACC4抗体和含高滴度的抗瓜氨酸化蛋白抗体的类风湿性关节炎患者血清中的抗体均能结合至CIA小鼠关节炎症关节软骨周围的滑膜组织；成功构建不同种系小鼠类风湿性关节炎模型，其发病率和疾病严重程度无统计学差异（P>0.05）；液相芯片技术分析结果显示，小鼠血清中的抗瓜氨酸化抗体低表达或几乎不表达，但抗CII三螺旋结构多肽的抗体反应性较强。结论 轻度急性炎症反应不足以引起关节软骨蛋白瓜氨酸化，其特异性抗原表位的表达需要高强度的炎症反应。二型胶原诱导的小鼠关节炎模型中炎症关节软骨蛋白可表达瓜氨酸化抗原表位，但瓜氨酸化抗原表位的表达不足以引起小鼠体内抗瓜氨酸化抗体的免疫应答，其免疫应答需要更多的刺激信号来诱导。

Abstract: Objective To investigate the expression of citrullinated epitopes in articular cartilage protein and whether its expression is sufficient to induce anti-citrullinated protein antibody (ACPA) response in mice. Methods The experimental group was treated with different concentrations of lipopolysaccharide (LPS), heat-inactivated bacteria (Escherichia coli and Staphylococcus aureus) or specific monoclonal antibody against type II collagen to induce citrullination of articular cartilage protein, with PBS as the control. Immunohistochemistry with the monoclonal antibody ACC4 (IgG1) that specifically binds to the citrullinated epitope of cartilage protein was performed for detecting the expression of citrullinated protein, with ACC1 (IgG2a) as a positive control antibody and L243 (IgG2a) and Hy2.15 (IgG1) as the negative isotype control. In the in vivo experiment, SD rats were subjected to injection of different doses of LPS in the right knee (with PBS as the controls in the left knee), and 3 days later frozen sections were prepared for immunohistochemical detection of the expression of citrullinated protein. Models of collagen-induced arthritis (CIA) established in different mouse strains were observed for incidence and severity of CIA. Serum samples collected from these models and the sera from rheumatoid arthritis patients were examined for anti-citrullinated protein antibody, and immunohistochemistry was performed to detect the expression of citrullinated protein in the cartilage of the mouse. Results The citrullinated CII epitope-specific antibody ACC4 did not bind to articular cartilage tissues with different treatments as compared with the positive control antibody ACC1. The ACC4 antibody and the antibodies from patients with rheumatoid arthritis with high titers of anti-citrullinated protein antibody were capable of binding to the synovial tissue around the articular cartilage of the CIA. Luminex analysis showed that the anti-citrullinated protein antibody was lowly expressed in mouse serum, but the anti-type II collagen triple helix structure peptide antibody exhibited strong reactivity. Conclusion Mild acute inflammatory response is not enough to cause citrullination of articular cartilage protein, and the expression of specific epitope requires a high-intensity inflammatory response. Inflammatory articular cartilage protein can express citrullinated epitopes in type II collagen-induced arthritis in mice, but the expression of citrullinated epitopes is not sufficient to induce an immune response to anti-citrullinated antibodies. Stronger stimulation signals are required to induce an immune response for producing anti-citrullinated protein antibodies.