Putative anti-muscarinic antibodies cannot be detected in patients with primary Sjogren's syndrome using conventional immunological approaches
Putative anti-muscarinic antibodies cannot be detected in patients with primary Sjogren's syndrome using conventional immunological approaches
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DOI：
10.1093/rheumatology/keh389
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发表时间：
2004-12-01
期刊：
影响因子：
5.5
通讯作者：
Smith, PM
中科院分区：
文献类型：
--
作者：
Dawson, LJ;Allison, HE;Smith, PM
Objective.为了确定是否针对毒蕈碱M3受体的自身抗体存在于原发性干燥综合征（pSS）患者的血清中，如果是这样，这些自身抗体是否抑制完整的唾液腺泡细胞的分泌。用蛋白G从15例pSS患者收集的血清中通过亲和层析纯化IgG。抗体检测采用蛋白质印迹法、全细胞酶联免疫吸附试验（ELISA）和免疫印迹法。使用Fura-2显微荧光法测定人和小鼠唾液腺腺泡细胞和转染人M3受体（CHO-M3）的中国仓鼠卵巢（CHO）细胞中细胞内Ca 2+活性（[Ca 2 +]（i））的变化，测定IgG片段的抗分泌活性。我们没有发现特异性M3受体识别的IgG组分从pSS患者使用蛋白质印迹或ELISA或免疫印迹技术，其中表位构象被保留。慢性暴露于pSS IgG对激动剂诱发的Ca 2+信号没有影响，在人或小鼠下颌下腺泡细胞或CHO-M3细胞中测量。然而，干燥综合征患者IgG的急性应用产生了激动剂刺激的[Ca 2 +]（i）升高的快速可逆性降低。这些数据代表pSS IgG对唾液腺泡细胞抑制的首次证明;然而，发现这种抑制是可逆的。我们的数据还表明，pSS IgG与M3 R的结合无法通过常规免疫学方法可视化。
Objective. To determine whether autoantibodies directed against muscarinic M3 receptors are present in the serum of patients with primary Sjogren's syndrome (pSS), and if so whether these autoantibodies inhibit secretion by intact salivary acinar cells.Methods. IgG was purified by affinity chromatography using protein G from sera collected from 15 patients with pSS. Antibody detection was by Western blotting, whole-cell enzyme-linked immunsorbent assay (ELISA) and immunoblotting. The antisecretory activity of the IgG faction was determined using fura-2 microfluorimetry to measure changes in intracellular Ca2+ activity ([Ca2+](i)) in human and mouse salivary gland acinar cells and in Chinese hamster ovary (CHO) cells transfected with human M3 receptors (CHO-M3).Results. We found no specific M3 receptor recognition by the IgG fraction obtained from pSS patients using either Western blotting or ELISA or immunoblot techniques in which epitope conformation were preserved. Chronic exposure to pSS IgG had no effect on agonist-evoked Ca2+ signals measured in human or mouse submandibular acinar cells or in CHO-M3 cells. However, acute application of IgG from Sjogren's syndrome patients produced a rapidly reversible reduction in the agonist-stimulated elevation in [Ca2+](i).Conclusion. These data represent the first demonstration of salivary acinar cell inhibition by pSS IgG; however, this inhibition was found to be reversible. Our data also show that pSS IgG binding to M3R cannot be visualized by conventional immunological approaches.