Böcker Ulrich, Yezerskyy Oleksandr, Feick Peter, Manigold Tobias, Panja Asit, Kalina Uwe, Herweck Frank, Rossol Siegbert, Singer Manfred V
Department of Medicine II and Medical Research Center, University of Heidelberg, Medical Faculty of Mannheim, Theodor-Kutzer-Ufer, 68167 Mannheim, Germany.
Int J Colorectal Dis. 2003 Jan;18(1):25-32. doi: 10.1007/s00384-002-0415-6. Epub 2002 Jun 14.
Luminal bacteria have been implicated in the pathogenesis of inflammatory bowel diseases. Exposure of intestinal epithelial cells (IEC) to bacterial components potentially initiates intestinal inflammation by release of chemokines and recruitment of inflammatory cells. We analyzed receptor expression and signaling pathways involved in activation of human primary IEC and carcinoma-derived cell lines by lipopolysaccharide (LPS).
HT-29/p, HT-29/MTX, and Caco-2 cells were stimulated by LPS. IL-8 content in supernatants was analyzed by ELISA, and expression of CD14, Toll-like receptor (TLR) 2 and TLR 4 was determined by RT-PCR. Presence of TLR 4 protein was assessed by western blot analysis. LPS response was modulated by sCD14, LPS-binding protein, neutralization of CD14, and inhibitors of early signal activation.
LPS dose-dependently induced secretion of IL-8 in undifferentiated HT-29/p cells while Caco-2 and permanently differentiated HT-29/MTX cells were unresponsive. Differently to HT-29/MTX, both HT-29/p and Caco-2 cells constitutively expressed transcripts for CD14. However, CD14 was not required for LPS-mediated induction of IL-8 in HT-29/p cells since neutralizing anti-CD14 antibodies left IL-8 levels unchanged. Unresponsiveness of Caco-2 and HT-29/MTX cells to LPS persisted in the presence of sCD14 and/or LPS-binding protein. Neither cell line expressed TLR 2 transcripts while only responsive HT-29/p cells expressed TLR 4 mRNA and TLR 4 protein. Butyrate down-regulated TLR 4 expression and significantly diminished LPS-dependent IL-8 secretion. Inhibition of G protein dependent kinase activation reduced IL-8 levels to 50%; the phosphatidyl-inositol-3'-kinase inhibitor LY294002 abrogated the response.
Responsiveness of IEC lines to LPS is positively correlated with TLR 4 expression. Strategies targeting TLR 4 expression or TLR 4 mediated signaling may antagonize IEC activation by LPS.
管腔细菌与炎症性肠病的发病机制有关。肠道上皮细胞（IEC）暴露于细菌成分可能通过趋化因子的释放和炎症细胞的募集引发肠道炎症。我们分析了脂多糖（LPS）激活人原代IEC和癌衍生细胞系所涉及的受体表达和信号通路。
用LPS刺激HT-29/p、HT-29/MTX和Caco-2细胞。通过ELISA分析上清液中的IL-8含量，通过RT-PCR测定CD14、Toll样受体（TLR）2和TLR 4的表达。通过蛋白质印迹分析评估TLR 4蛋白的存在。通过sCD14、LPS结合蛋白、CD14中和以及早期信号激活抑制剂调节LPS反应。
LPS剂量依赖性地诱导未分化的HT-29/p细胞分泌IL-8，而Caco-2和永久分化型HT-29/MTX细胞无反应。与HT-29/MTX不同，HT-29/p和Caco-2细胞均组成性表达CD14转录本。然而，在HT-29/p细胞中，LPS介导的IL-8诱导不需要CD14，因为中和抗CD14抗体后IL-8水平不变。在存在sCD14和/或LPS结合蛋白的情况下，Caco-2和HT-29/MTX细胞对LPS仍无反应。两种细胞系均不表达TLR 2转录本，而只有有反应的HT-29/p细胞表达TLR 4 mRNA和TLR 4蛋白。丁酸盐下调TLR 4表达并显著减少LPS依赖性IL-8分泌。抑制G蛋白依赖性激酶激活可使IL-8水平降至50%；磷脂酰肌醇-3'-激酶抑制剂LY294002消除了反应。
IEC系对LPS的反应性与TLR 4表达呈正相关。靶向TLR 4表达或TLR 4介导信号传导的策略可能拮抗LPS对IEC的激活作用。
