Sisto Margherita, D'Amore Massimo, Caprio Simone, Mitolo Vincenzo, Scagliusi Pasquale, Lisi Sabrina
Department of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy.
Ann N Y Acad Sci. 2009 Aug;1171:407-14. doi: 10.1111/j.1749-6632.2009.04688.x.
Inhibition of tumor necrosis factor-alpha (TNF-alpha) in organ-specific autoimmune disease is proving efficacious for a large number of patients. A wide array of biological agents has been designed to inhibit TNF-alpha, such as adalimumab (fully humanized) and etanercept (soluble TNF-alpha receptor fusion constructs p75 subunit). Recently, we suggested that anti-Ro and anti-La autoantibodies (Abs) isolated from patients with Sjögren's syndrome, an autoimmune rheumatic disease, are able to trigger cell death through extrinsic apoptotic mechanisms in human salivary gland epithelial cells (SGEC). We analyzed if primary human SGEC cultures, established from biopsy of labial minor salivary glands, are able to produce TNF-alpha, an inductor of the extrinsic apoptotic pathway, when treated with anti-Ro autoantibodies. A comparative study was performed to test the efficacy of adalimumab and etanercept to block TNF-alpha-mediated apoptosis. ELISA assay and RT-PCR were employed to visualize TNF-alpha production, and apoptosis was evaluated by DNA ladder and flow cytometry. We found that cell treatment with anti-Ro autoantibodies determines TNF-alpha production that reaches a maximum at 16 h and is decreased (P < 0.05) at 24 and 48 h. Adalimumab seems to be more efficacious than etanercept in blocking TNF-alpha-mediated apoptosis. The YOPRO-1 (+) and propidium iodide (-) method revealed 60% of apoptotic cells after 24 h of incubation with anti-Ro compared with 15% of apoptotic cells treated with anti-Ro plus adalimumab and 25% of apoptotic cells treated with anti-Ro plus etanercept. The antiapoptotic effect of adalimumab and etanercept was supported by inhibition of DNA laddering induced by anti-Ro Abs. These data validate the therapeutic efficacy of the anti-TNF reagents in the treatment of autoimmune disorders.
在器官特异性自身免疫性疾病中，抑制肿瘤坏死因子-α（TNF-α）已被证明对大量患者有效。已经设计了多种生物制剂来抑制TNF-α，例如阿达木单抗（完全人源化）和依那西普（可溶性TNF-α受体融合构建体p75亚基）。最近，我们发现从自身免疫性风湿性疾病干燥综合征患者中分离出的抗Ro和抗La自身抗体（Abs）能够通过外源性凋亡机制触发人唾液腺上皮细胞（SGEC）的细胞死亡。我们分析了从唇小唾液腺活检建立的原代人SGEC培养物在用抗Ro自身抗体处理时是否能够产生TNF-α，TNF-α是外源性凋亡途径的诱导剂。进行了一项比较研究，以测试阿达木单抗和依那西普阻断TNF-α介导的细胞凋亡的功效。采用ELISA测定法和RT-PCR来观察TNF-α的产生，并通过DNA梯状条带和流式细胞术评估细胞凋亡。我们发现用抗Ro自身抗体处理细胞可导致TNF-α的产生，在16小时时达到最大值，在24小时和48小时时降低（P <0.05）。在阻断TNF-α介导的细胞凋亡方面，阿达木单抗似乎比依那西普更有效。YOPRO-1（+）和碘化丙啶（-）方法显示，与抗Ro加阿达木单抗处理的15%凋亡细胞和抗Ro加依那西普处理的25%凋亡细胞相比，在用抗Ro孵育24小时后，60%的细胞发生凋亡。阿达木单抗和依那西普的抗凋亡作用得到了抗Ro抗体诱导的DNA梯状条带抑制的支持。这些数据证实了抗TNF试剂在治疗自身免疫性疾病方面的治疗效果。
