Department of Immunology Research, Centocor Research and Development, Inc., Radnor, PA 19087, USA.
Nephron Exp Nephrol. 2011;117(4):e114-23. doi: 10.1159/000320177. Epub 2010 Oct 2.
BACKGROUND/AIMS: Interleukin-17A (IL-17A) is a T cell-derived inflammatory cytokine that is upregulated during renal allograft rejection. The present study sought to further describe the IL-17A-mediated proinflammatory/profibrotic activity of proximal tubule epithelium that may contribute to allograft rejection.
Immortalized (HK-2) and primary (HRPTEpiC) human proximal tubule epithelial cells were utilized for this study. Profibrotic gene alterations were examined by real-time quantitative PCR. Inflammatory mediator secretion was examined by multiplex bead-based detection of secreted proteins. Immunofluorescence microscopy and immunoblotting were utilized to examine alterations in junctional protein expression and cell morphology.
In HK-2 cells IL-17A significantly downregulated the expression of the proepithelial gene CDH1 (E-cadherin) while the proinflammatory/profibrotic genes CTGF, CD44 and TGFBR1 were significantly increased. IL-17A also increased the secretion of fractalkine, G-CSF, GM-CSF, VEGF, IL-6 and IL-8. In HRPTEpiC 100 ng/ml IL-17A upregulated the proinflammatory/profibrotic genes ACTA2, CCL2, CHMP1A, CTGF, FN1, IL6, FSP1, SMAD1, SMAD5, TGFB1 and TGFBR2 while treatment with a reduced concentration of IL-17A (0.1 ng/ml) decreased SMAD5, TGFB1 and PDGFRB expression. Changes in ZO-1 and E-cadherin protein expression and cell morphology were examined following IL-17A treatment as indicators of epithelial-to-mesenchymal transition. IL-17A decreased ZO-1 expression in HK-2 and HRPTEpiC; however, E-cadherin was only reduced in HK-2 cells. Neither HK-2 nor HRPTEpiC assumed an elongated, fibroblast-like morphology following IL-17A treatment.
IL-17A directly mediates proximal tubule epithelial cell proinflammatory/profibrotic activity as demonstrated by the alteration in genes associated with extracellular matrix remodeling and cell-cell interaction, and stimulation of inflammatory mediator and immune cell chemoattractant secretion. Additionally, IL-17A may have a negative impact on barrier integrity as indicated by ZO-1 downregulation.
背景/目的：白细胞介素-17A（IL-17A）是一种由 T 细胞衍生的炎症细胞因子，在肾移植排斥反应中上调。本研究旨在进一步描述近端肾小管上皮细胞中 IL-17A 介导的促炎/促纤维化活性，这可能有助于移植物排斥反应。
本研究采用永生化（HK-2）和原代（HRPTEpiC）人近端肾小管上皮细胞。通过实时定量 PCR 检测促纤维化基因改变。通过基于多重珠的分泌蛋白检测来检测炎症介质的分泌。免疫荧光显微镜和免疫印迹用于检测连接蛋白表达和细胞形态的改变。
在 HK-2 细胞中，IL-17A 显著下调了上皮基因 CDH1（E-钙粘蛋白）的表达，而促炎/促纤维化基因 CTGF、CD44 和 TGFBR1 的表达显著增加。IL-17A 还增加了 fractalkine、G-CSF、GM-CSF、VEGF、IL-6 和 IL-8 的分泌。在 HRPTEpiC 中，100ng/ml IL-17A 上调了促炎/促纤维化基因 ACTA2、CCL2、CHMP1A、CTGF、FN1、IL6、FSP1、SMAD1、SMAD5、TGFB1 和 TGFBR2，而用较低浓度的 IL-17A（0.1ng/ml）处理则降低了 SMAD5、TGFB1 和 PDGFRB 的表达。用 IL-17A 处理后，通过检测上皮-间充质转化的标志物 ZO-1 和 E-钙粘蛋白蛋白表达和细胞形态的变化，来研究细胞形态的变化。IL-17A 降低了 HK-2 和 HRPTEpiC 中的 ZO-1 表达，但仅在 HK-2 细胞中降低了 E-钙粘蛋白的表达。用 IL-17A 处理后，HK-2 和 HRPTEpiC 都没有呈现出伸长的成纤维细胞样形态。
IL-17A 通过改变与细胞外基质重塑和细胞间相互作用相关的基因，刺激炎症介质和免疫细胞趋化因子的分泌，直接介导近端肾小管上皮细胞的促炎/促纤维化活性。此外，ZO-1 的下调表明 IL-17A 可能对屏障完整性产生负面影响。
