Hong Chien-Hui, Chang Kee-Lung, Wang Hung-Jen, Yu Hsin-Su, Lee Chih-Hung
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Dermatology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan; Department of Dermatology, National Yang-Ming University College of Medicine, Taipei, Taiwan.
Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
J Dermatol Sci. 2015 Jun;78(3):206-14. doi: 10.1016/j.jdermsci.2015.03.004. Epub 2015 Mar 16.
IL-9 and its receptor play important roles in the pathogenesis of asthma. Its role in atopic dermatitis (AD) was examined in just a few studies, including nucleotide polymorphisms, increased transcriptional levels of IL-9 and IL-9R in diseased skin, and an association of blood IL-9 levels with clinical severity.
Little was known about the pathophysiological regulation of IL-9/IL-9R in AD skin. We asked whether IL-9R was expressed in epidermal keratinocytes; if so, what the functional outcome, cytokine production, and signaling pathway of IL-9/IL-9R in keratinocytes are.
We measured and compared the expression of IL-9R in skin from AD patients and controls by immunofluorescence. We also performed in vitro studies on the IL-9-treated primary keratinocytes, including flow cytometry for IL-9R expressions, Western blotting for mTOR, S6K, ERK, p38, and STAT3 activations, ELISA for cytokine levels, and immunofluorescence for STIM1.
We found that IL-9R was indeed expressed in keratinocytes but not in fibroblasts. Its expression in keratinocytes was enhanced by IL-4 but not by TGF-beta1. IL-9 induced a moderate production of IL-8 but not CXCL16, CCL22, TSLP, nor IL-33. IL-9 induced formation of STIM1-puncta. IL-9 induced ERK phosphorylation both dose- and time-dependently, but not mTOR, S6K, p38, or STAT3. Pretreatment with U0126 (ERK inhibitor) but not rapamycin (mTOR inhibitor) abrogated the IL-9-mediated IL-8 production. Blockage of STIM1 with BTP2 or SKF96265 abrogated ERK phosphorylation and IL-8 production induced by IL-9.
This study represents the first to show the regulation of the IL-9-STIM1-ERK-IL-8 axis in keratinocyte, and how the axis might play an important role in the pathophysiology of AD.
白细胞介素-9（IL-9）及其受体在哮喘发病机制中起重要作用。关于其在特应性皮炎（AD）中的作用仅在少数研究中有所探讨，包括核苷酸多态性、病变皮肤中IL-9和IL-9受体转录水平升高以及血液中IL-9水平与临床严重程度的关联。
关于AD皮肤中IL-9/IL-9受体的病理生理调节知之甚少。我们研究IL-9受体是否在表皮角质形成细胞中表达；如果是，角质形成细胞中IL-9/IL-9受体的功能结果、细胞因子产生及信号通路是什么。
我们通过免疫荧光测量并比较AD患者和对照皮肤中IL-9受体的表达。我们还对经IL-9处理的原代角质形成细胞进行了体外研究，包括用于IL-9受体表达的流式细胞术、用于mTOR、S6K、ERK、p38和STAT3激活的蛋白质印迹法、用于细胞因子水平的酶联免疫吸附测定法以及用于基质相互作用分子1（STIM1）的免疫荧光法。
我们发现IL-9受体确实在角质形成细胞中表达，但在成纤维细胞中不表达。其在角质形成细胞中的表达受IL-4增强，但不受转化生长因子-β1（TGF-β1）增强。IL-9诱导适度产生IL-8，但不诱导CXCL16、CCL22、胸腺基质淋巴细胞生成素（TSLP）或IL-33。IL-9诱导STIM1斑点形成。IL-9剂量和时间依赖性地诱导ERK磷酸化，但不诱导mTOR、S6K、p38或STAT3磷酸化。用U0126（ERK抑制剂）而非雷帕霉素（mTOR抑制剂）预处理可消除IL-9介导的IL-8产生。用BTP2或SKF96265阻断STIM1可消除IL-9诱导的ERK磷酸化和IL-8产生。
本研究首次表明角质形成细胞中IL-9-STIM1-ERK-IL-8轴的调节，以及该轴可能在AD病理生理学中发挥的重要作用。
