Kinloch Andrew, Lundberg Karin, Wait Robin, Wegner Natalia, Lim Ngee Han, Zendman Albert J W, Saxne Tore, Malmström Vivianne, Venables Patrick J
Imperial College London, London, UK.
Arthritis Rheum. 2008 Aug;58(8):2287-95. doi: 10.1002/art.23618.
To examine synovial fluid as a site for generating citrullinated antigens, including the candidate autoantigen citrullinated alpha-enolase, in rheumatoid arthritis (RA).
Synovial fluid was obtained from 20 patients with RA, 20 patients with spondylarthritides (SpA), and 20 patients with osteoarthritis (OA). Samples were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by staining with Coomassie blue and immunoblotting for citrullinated proteins, alpha-enolase, and the deiminating enzymes peptidylarginine deiminase type 2 (PAD-2) and PAD-4. Proteins from an RA synovial fluid sample were separated by 2-dimensional electrophoresis, and each protein was identified by immunoblotting and mass spectrometry. Antibodies to citrullinated alpha-enolase peptide 1 (CEP-1) and cyclic citrullinated peptide 2 were measured by enzyme-linked immunosorbent assay.
Citrullinated polypeptides were detected in the synovial fluid from patients with RA and patients with SpA, but not in OA samples. Alpha-enolase was detected in all of the samples, with mean levels of 6.4 ng/microl in RA samples, 4.3 ng/microl in SpA samples, and <0.9 ng/microl in OA samples. Two-dimensional electrophoresis provided evidence that the alpha-enolase was citrullinated in RA synovial fluid. The citrullinating enzyme PAD-4 was detected in samples from all 3 disease groups. PAD-2 was detected in 18 of the RA samples, in 16 of the SpA samples, and in none of the OA samples. Antibodies to CEP-1 were found in 12 of the RA samples (60%), in none of the SpA samples, and in 1 OA sample.
These results highlight the importance of synovial fluid for the expression of citrullinated autoantigens in inflammatory arthritis. Whereas the expression of citrullinated proteins is a product of inflammation, the antibody response remains specific for RA.
研究类风湿关节炎（RA）患者的滑液是否为瓜氨酸化抗原（包括候选自身抗原瓜氨酸化α-烯醇化酶）的产生部位。
收集20例RA患者、20例脊柱关节炎（SpA）患者和20例骨关节炎（OA）患者的滑液样本。样本经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离，考马斯亮蓝染色，然后进行瓜氨酸化蛋白、α-烯醇化酶以及脱氨基酶肽基精氨酸脱亚氨酶2型（PAD-2）和肽基精氨酸脱亚氨酶4型（PAD-4）的免疫印迹检测。对一份RA滑液样本中的蛋白质进行二维电泳分离，通过免疫印迹和质谱鉴定每种蛋白质。采用酶联免疫吸附测定法检测抗瓜氨酸化α-烯醇化酶肽1（CEP-1）和环瓜氨酸肽2的抗体。
在RA患者和SpA患者的滑液中检测到瓜氨酸化多肽，但OA样本中未检测到。所有样本中均检测到α-烯醇化酶，RA样本中的平均水平为6.4 ng/μl，SpA样本中的平均水平为4.3 ng/μl，OA样本中的平均水平<0.9 ng/μl。二维电泳结果表明，RA滑液中的α-烯醇化酶发生了瓜氨酸化。在所有3个疾病组的样本中均检测到瓜氨酸化酶PAD-4。在18份RA样本、16份SpA样本中检测到PAD-2，OA样本中均未检测到。12份RA样本（60%）中检测到抗CEP-1抗体，SpA样本中均未检测到，1份OA样本中检测到抗CEP-1抗体。
这些结果突出了滑液在炎性关节炎中瓜氨酸化自身抗原表达中的重要性。虽然瓜氨酸化蛋白的表达是炎症的产物，但抗体反应仍对RA具有特异性。
