Ling Song, Lai Angela, Borschukova Olga, Pumpens Paul, Holoshitz Joseph
University of Michigan, Ann Arbor 48109-0680, USA.
Arthritis Rheum. 2006 Nov;54(11):3423-32. doi: 10.1002/art.22178.
Susceptibility to rheumatoid arthritis (RA) is closely associated with HLA-DRB1 alleles encoding a shared epitope (SE) in positions 70-74 of the HLA-DRbeta chain. The mechanistic basis for this association is unknown. Given the proposed pathogenic role of nitric oxide (NO) in RA, this study was undertaken to examine whether the SE can trigger NO signaling events.
The intracellular levels of NO were measured with the fluorescent NO probe 4,5-diaminofluorescein diacetate and by the 2,3-diaminonaphthalene method. NO synthase activity was determined by measuring the rate of conversion of radioactive arginine to citrulline. Levels of cGMP were measured with a commercial enzyme-linked immunosorbent assay, and the cytolytic activity of T cells was measured using a standard (51)Cr release assay.
Lymphoblastoid B cell lines carrying SE-positive HLA-DR alleles displayed a higher rate of spontaneous NO production compared with SE-negative cells. L cell transfectants expressing SE-positive DR molecules on their surface also generated higher levels of NO. Tetrameric HLA-DR molecules containing a DRbeta-chain encoded by the SE-positive DRB10401 allele stimulated fibroblast cells to produce higher levels of NO compared with cells stimulated with a control HLA-DR tetramer. Multimeric hepatitis B core proteins engineered to express region 65-79 encoded by the DRB10401 allele, but not the same region encoded by the control allele DRB1*0402, stimulated NO production in fibroblasts. Similarly, synthetic 15-mer peptides corresponding to the region 65-79 encoded by SE-positive alleles triggered increased NO levels when incubated with class II major histocompatibility complex-negative cells. The signaling pathway was found to involve NO synthase activation, followed by increased production of cGMP. SE-triggered increased NO levels inhibited cytolytic elimination of target cells.
The SE can trigger NO-mediated signaling events in opposite cells, and may thereby contribute to RA pathogenesis.
类风湿关节炎（RA）易感性与在HLA - DRβ链70 - 74位编码共享表位（SE）的HLA - DRB1等位基因密切相关。这种关联的机制基础尚不清楚。鉴于一氧化氮（NO）在RA中所提出的致病作用，本研究旨在检测SE是否能触发NO信号事件。
用荧光NO探针4,5 - 二氨基荧光素二乙酸酯并通过2,3 - 二氨基萘法测量细胞内NO水平。通过测量放射性精氨酸向瓜氨酸的转化速率来测定NO合酶活性。用商业酶联免疫吸附测定法测量cGMP水平，并用标准的（51）Cr释放测定法测量T细胞的细胞溶解活性。
与SE阴性细胞相比，携带SE阳性HLA - DR等位基因的淋巴母细胞样B细胞系显示出自发NO产生率更高。在其表面表达SE阳性DR分子的L细胞转染子也产生更高水平的NO。与用对照HLA - DR四聚体刺激的细胞相比，含有由SE阳性DRB10401等位基因编码的DRβ链的四聚体HLA - DR分子刺激成纤维细胞产生更高水平的NO。经工程改造以表达由DRB10401等位基因编码的65 - 79区域而非对照等位基因DRB1*0402编码的相同区域的多聚体乙型肝炎核心蛋白刺激成纤维细胞产生NO。同样，与II类主要组织相容性复合体阴性细胞孵育时，对应于SE阳性等位基因编码的65 - 79区域的合成15聚体肽触发NO水平升高。发现该信号通路涉及NO合酶激活，随后cGMP产生增加。SE触发的NO水平升高抑制了靶细胞的细胞溶解清除。
SE可在异体细胞中触发NO介导的信号事件，从而可能有助于RA发病机制。
