Chernyavsky Alex, Chen Yumay, Wang Ping H, Grando Sergei A
Department of Dermatology, University of California Irvine, CA, USA.
Department of Medicine, University of California Irvine, CA, USA.
Int Immunopharmacol. 2015 Nov;29(1):76-80. doi: 10.1016/j.intimp.2015.04.046. Epub 2015 May 18.
The mechanism of detachment and death of keratinocytes in pemphigus vulgaris (PV) involves pro-apoptotic action of constellations of autoantibodies determining disease severity and response to treatment. The presence of antibodies to nicotinic acetylcholine receptors (nAChRs) and the therapeutic efficacy of cholinomimetics in PV is well-established. Recently, adsorption of anti-mitochondrial antibodies abolished the ability of PVIgGs to cause acantholysis, demonstrating their pathophysiological significance. Since, in addition to cell membrane, nAChRs are also present on the mitochondrial outer membrane, wherein they act to prevent activation of intrinsic (mitochondrial apoptosis), we hypothesized that mitochondrial (mt)-nAChRs might be targeted by PVIgGs. To test this hypothesis, we employed the immunoprecipitation-western blot assay of keratinocyte mitochondrial proteins that visualized the α3, α5, α7, α9, α10, β2 and β4 mt-nAChR subunits precipitated by PV IgGs, suggesting that functions of mt-nAChRs are compromised in PV. To pharmacologically counteract the pro-apoptotic action of anti-mitochondrial antibodies in PV, we exposed naked keratinocyte mitochondria to PVIgGs in the presence of the nicotinic agonist nicotine ± antagonists, and measured cytochrome c (CytC) release. Nicotine abolished PVIgG-dependent CytC release, showing a dose-dependent effect, suggesting that protection of mitochondria can be a novel mechanism of therapeutic action of nicotinic agonists in PV. The obtained results indicated that the mt-nAChRs targeted by anti-mitochondrial antibodies produced by PV patients are coupled to inhibition of CytC release, and that nicotinergic stimulation can abolish PVIgG-dependent activation of intrinsic apoptosis in KCs. Future studies should determine if and how the distinct anti-mt-nAChR antibodies penetrate KCs and correlate with disease severity.
寻常型天疱疮（PV）中角质形成细胞的分离和死亡机制涉及自身抗体组合的促凋亡作用，这些自身抗体决定了疾病的严重程度和对治疗的反应。抗烟碱型乙酰胆碱受体（nAChRs）抗体的存在以及拟胆碱药在PV中的治疗效果已得到充分证实。最近，抗线粒体抗体的吸附消除了PV免疫球蛋白G（PVIgGs）引起棘层松解的能力，证明了它们的病理生理意义。由于除细胞膜外，nAChRs也存在于线粒体外膜上，在那里它们起到防止内在（线粒体凋亡）激活的作用，我们推测线粒体（mt）-nAChRs可能是PVIgGs的作用靶点。为了验证这一假设，我们采用角质形成细胞线粒体蛋白的免疫沉淀-蛋白质印迹分析，该分析可视化了由PV免疫球蛋白G沉淀的α3、α5、α7、α9、α10、β2和β4 mt-nAChR亚基，表明PV中mt-nAChRs的功能受到损害。为了从药理学上对抗PV中抗线粒体抗体的促凋亡作用，我们在存在烟碱激动剂尼古丁±拮抗剂的情况下，将裸露的角质形成细胞线粒体暴露于PVIgGs中，并测量细胞色素c（CytC）释放。尼古丁消除了PVIgG依赖的CytC释放，呈剂量依赖性效应，表明保护线粒体可能是烟碱激动剂在PV中治疗作用的新机制。获得的结果表明，PV患者产生的抗线粒体抗体靶向的mt-nAChRs与抑制CytC释放有关，并且烟碱能刺激可以消除PVIgG依赖的角质形成细胞中内在凋亡的激活。未来的研究应确定不同的抗mt-nAChR抗体是否以及如何穿透角质形成细胞并与疾病严重程度相关。
