Joshua Vijay, Schobers Loes, Titcombe Philip J, Israelsson Lena, Rönnelid Johan, Hansson Monika, Catrina Anca I, Pruijn Ger J M, Malmström Vivianne
Rheumatology Unit, Department of Medicine, Karolinska Institute, Karolinska University Hospital Solna, 17176, Stockholm, Sweden.
Department of Biomolecular Chemistry, Radboud Institute for Molecular Life Sciences and Institute for Molecules and Materials, Radboud University, Nijmegen, Netherlands.
Arthritis Res Ther. 2016 Dec 1;18(1):284. doi: 10.1186/s13075-016-1181-0.
Antibodies against citrullinated proteins (ACPA) are common in patients with rheumatoid arthritis (RA). ACPA can appear before disease onset and target many self-antigens. Citrullinated fibrin/fibrinogen represents a classical ACPA target antigen, and mass spectrometry of RA synovial fluid reveals elevated citrullinated (cit) fibrinogen (Fib) peptides compared to non-RA controls. We investigated the extent to which these less-studied peptides represent autoantibody targets and sought to visualize the corresponding cit-Fib-reactive B cells in RA patients.
An in-house ELISA was established against four cit-Fib α-subunit peptides (cit-Fib α-35; cit-Fib α-216,218; cit-Fib α-263,271 and cit-Fib α-425,426) and serum from patients with established RA (n = 347) and disease controls with psoriatic arthritis (PsA) or ankylosing spondylitis (AS) (n = 236) were analyzed. RA patients were genotyped for HLA-DR alleles, PTPN22 R620W and screened for anti-CCP2 and cit-Fib protein antibodies. The cit-Fib peptides were also used to assemble antigen tetramers to identify cit-Fib-reactive B cells in peripheral blood by flow cytometry.
The frequencies of autoantibodies against different cit-Fib epitopes in RA patients compared to PsA/AS patients were: cit-Fib α-35 (RA 20%, vs PsA/AS 1%); cit-Fib α-216,218 (13% vs 0.5%); cit-Fib α-263,271 (21% vs 0.5%) and cit-Fib α-425,426 (17% vs 1%). The presence of autoantibodies against these peptides was associated with presence of anti-CCP2 and anti-cit-Fib protein antibodies. No association was found between HLA-DR shared epitope and antibodies to the different cit-Fib peptides. However, association was observed between the PTPN22 risk allele and positivity to cit-Fib α-35 and cit-Fib α-263,271. B cells carrying surface Ig reactive to these cit-Fib peptides were found in RA peripheral blood and these tend to be more common in PTPN22 risk allele carriers.
Our data show that several cit-Fib peptides are targeted by autoantibodies in RA, but not in PsA/AS, implicating that these are not due to arthritis but more specific for RA etiology. The RA-associated anti-cit protein response is broad with many parallel immune responses. The association between cit-Fib autoantibodies and the PTPN22 R620W risk allele supports the hypothesis of altered B cell regulation, such as autoreactive B cells evading tolerance checkpoints.
抗瓜氨酸化蛋白抗体（ACPA）在类风湿关节炎（RA）患者中很常见。ACPA可在疾病发作前出现，并靶向多种自身抗原。瓜氨酸化纤维蛋白/纤维蛋白原是一种典型的ACPA靶抗原，与非RA对照相比，RA滑液的质谱分析显示瓜氨酸化（cit）纤维蛋白原（Fib）肽水平升高。我们研究了这些研究较少的肽作为自身抗体靶标的程度，并试图在RA患者中可视化相应的cit-Fib反应性B细胞。
建立了针对四种cit-Fibα亚基肽（cit-Fibα-35；cit-Fibα-216,218；cit-Fibα-263,271和cit-Fibα-425,426）的内部ELISA，并分析了确诊RA患者（n = 347）以及银屑病关节炎（PsA）或强直性脊柱炎（AS）疾病对照患者（n = 236）的血清。对RA患者进行HLA-DR等位基因、PTPN22 R620W基因分型，并检测抗CCP2和cit-Fib蛋白抗体。这些cit-Fib肽还用于组装抗原四聚体，通过流式细胞术鉴定外周血中cit-Fib反应性B细胞。
与PsA/AS患者相比，RA患者中针对不同cit-Fib表位的自身抗体频率为：cit-Fibα-35（RA为20%，vs PsA/AS为1%）；cit-Fibα-216,218（13% vs 0.5%）；cit-Fibα-263,271（21% vs 0.5%）和cit-Fibα-425,426（17% vs 1%）。针对这些肽的自身抗体的存在与抗CCP2和抗cit-Fib蛋白抗体的存在相关。未发现HLA-DR共享表位与不同cit-Fib肽抗体之间存在关联。然而，观察到PTPN22风险等位基因与cit-Fibα-35和cit-Fibα-263,271阳性之间存在关联。在RA外周血中发现携带对这些cit-Fib肽有反应的表面Ig的B细胞，并且在PTPN22风险等位基因携带者中更常见。
我们的数据表明，几种cit-Fib肽是RA中自身抗体的靶标，但在PsA/AS中不是，这意味着这些不是由关节炎引起的，而是RA病因更具特异性。RA相关的抗瓜氨酸化蛋白反应广泛，有许多平行的免疫反应。cit-Fib自身抗体与PTPN22 R620W风险等位基因之间的关联支持B细胞调节改变的假说，例如自身反应性B细胞逃避耐受检查点。
