Horai Yoshiro, Nakamura Hideki, Nakashima Yoshikazu, Hayashi Tomayoshi, Kawakami Atsushi
a Unit of Translational Medicine, Department of Immunology and Rheumatology , Nagasaki University Graduate School of Biomedical Sciences, Sakamoto , Nagasaki , Japan.
b Department of Pathology , Nagasaki Prefectural Shimabara Hospital, Shimabara , Nagasaki , Japan.
Mod Rheumatol. 2016;26(1):99-104. doi: 10.3109/14397595.2015.1045256. Epub 2015 May 28.
The aim of this study was to clarify the molecular mechanisms elicited by toll-like receptor (TLR)3 in salivary gland cell death in patients with SS.
Expression of TLR3 and its downstream molecules was examined by immunohistochemical analysis, immunofluorescence, Western blot (WB), and antibody dot-blot array in labial salivary glands (LSGs), and cultured primary salivary gland epithelial cells (SGECs) obtained from patients with SS. We also investigated the difference of expression between ducts/alveoli of LSGs and cultured SGECs.
Phosphorylated Fas-associated protein with death domain (p-FADD) or caspase-8 was not found in ducts or alveoli of LSGs from SS patients and controls. Weak expression of receptor-interacting serine/threonine-protein kinase 3 (RIPK3) was found in SS patients, whereas no staining was observed in LSGs of controls. In contrast to LSGs, stimulation of SGECs with polyinosinic:cytidylic acid (poly I:C) significantly induced the expression of RIPK3, p-FADD, and cleaved caspase-8 by immunofluorescence and RIPK3, p-FADD, and cleaved caspase-3 by WB. However, it was counteracted by epidermal growth factor (EGF). Co-localization of anti-apoptotic molecules hemeoxygenase-2, heat shock protein 27, and p-protein kinase B or p-Akt was induced in EGF-stimulated SGECs.
We observed that poly I:C induced apoptosis of SGECs in vitro compared with a relatively low prevalence of apoptosis found in the ducts and alveoli of LSGs in vivo. Thus, we speculate that other counter-regulatory mechanisms, including those induced by EGF, might exist to protect against TLR3-mediated apoptosis of ductal and acinar epithelial cells in vivo.
本研究旨在阐明Toll样受体（TLR）3引发干燥综合征（SS）患者唾液腺细胞死亡的分子机制。
通过免疫组织化学分析、免疫荧光、蛋白质印迹法（WB）和抗体点杂交阵列检测唇腺（LSG）以及从SS患者获取的原代培养唾液腺上皮细胞（SGEC）中TLR3及其下游分子的表达。我们还研究了LSG的导管/腺泡与培养的SGEC之间的表达差异。
在SS患者和对照者的LSG导管或腺泡中未发现磷酸化的死亡结构域相关蛋白Fas（p-FADD）或半胱天冬酶-8。在SS患者中发现受体相互作用丝氨酸/苏氨酸蛋白激酶3（RIPK3）表达较弱，而在对照者的LSG中未观察到染色。与LSG不同，用聚肌苷酸:胞苷酸（poly I:C）刺激SGEC可通过免疫荧光显著诱导RIPK3、p-FADD和裂解的半胱天冬酶-8的表达，并通过WB诱导RIPK3、p-FADD和裂解的半胱天冬酶-3的表达。然而，它被表皮生长因子（EGF）抵消。在EGF刺激的SGEC中诱导了抗凋亡分子血红素加氧酶-2、热休克蛋白27和磷酸化蛋白激酶B或磷酸化Akt的共定位。
我们观察到与体内LSG导管和腺泡中相对较低的凋亡发生率相比，poly I:C在体外诱导了SGEC的凋亡。因此，我们推测可能存在其他反调节机制，包括那些由EGF诱导的机制，以保护体内导管和腺泡上皮细胞免受TLR3介导的凋亡。
