Induction of tolerance to lipopolysaccharide and mycobacterial components in Chinese hamster ovary/CD14 cells is not affected by overexpression of toll-like receptors 2 or 4
Induction of tolerance to lipopolysaccharide and mycobacterial components in Chinese hamster ovary/CD14 cells is not affected by overexpression of toll-like receptors 2 or 4
复制标题
DOI：
10.4049/jimmunol.167.4.2257
复制
发表时间：
2001-08-15
影响因子：
4.4
通讯作者：
Vogel, SN
中科院分区：
文献类型：
--
作者：
Medvedev, AE;Henneke, P;Vogel, SN
LPS刺激后Toll样受体（TLR）4的细胞表面表达下调已被认为是内毒素耐受的基础。在这项研究中，我们研究了TLR 2或TLR 4的过度表达是否会影响细胞对LPS或分枝杆菌组分阿拉伯糖加帽的脂阿拉伯甘露聚糖（LAM）和可溶性结核因子（STF）的耐受性。为此，用NF-κ B依赖性报告基因构建体内皮白细胞粘附分子CD 25（3E 10克隆）稳定转染的中国仓鼠卵巢/CD 14细胞被工程化以过表达人TLR 2或TLR 4。转染的TLR表现出适当的信号传导功能，如通过增加的3E 10/TLR 4细胞的LPS反应性和在将TLR 2转染到TLR 2阴性3E 10细胞中后获得对TLR 2特异性配体的敏感性所证明的。用LPS、LAM或STF预处理细胞不调节TLR 2或TLR 4细胞表面表达。LPS暴露后，3E 10、3E 10/TLR 2和3E 10/TLR 4细胞表现出LPS介导的NF-κ B活化和丝裂原活化蛋白（MAP）激酶磷酸化的相当的降低。同样，LPS预处理显著抑制LPS诱导的NF-κ B在同时过表达人TLR 4和髓样分化蛋白-2（MD-2）的中国仓鼠卵巢细胞中的易位，但未能调节TLR 4或MD-2细胞表面表达。用LAM或STF预处理3E 10/TLR 2细胞可降低随后用这些物质或LPS刺激诱导的NF-κ B反应。相反，3E 10/TLR 2细胞先前暴露于LPS导致对UPS、LAM和STF的低反应性，表明LPS和分枝杆菌产物诱导交叉耐受。因此，对LPS和分枝杆菌组分的耐受性不能仅仅归因于TLR/MD-2表达水平的降低，这表明抑制了其他信号传导中间体的表达或功能。
Down-regulation of cell surface expression of Toll-like receptor (TLR) 4 following LPS stimulation has been suggested to underlie endotoxin tolerance. In this study, we examined whether overexpression of TLR2 or TLR4 would affect the ability of cells to become tolerant to LPS or the mycobacterial components, arabinose-capped lipoarabinomannan (LAM) and soluble tuberculosis factor (STF). To this end, Chinese hamster ovary/CD14 cells stably transfected with a NF-kappaB-dependent reporter construct, endothelial leukocyte adhesion molecule CD25 (the 3E10 clone), were engineered to overexpress either human TLR2 or TLR4. Transfected TLRs exhibited proper signaling functions, as evidenced by increased LPS responsiveness of 3E10/TLR4 cells and acquisition of sensitivity to TLR2-specific ligands upon transfection of TLR2 into TLR2-negative 3E10 cells. Pretreatment of cells with LPS, LAM, or STF did not modulate TLR2 or TLR4 cell surface expression. Following LPS exposure, 3E10, 3E10/TLR2, and 3E10/TLR4 cells exhibited comparable decreases in LPS-mediated NF-kappaB activation and mitogen-activated protein (MAP) kinase phosphorylation. Likewise, LPS pretreatment profoundly inhibited LPS-induced NF-kappaB translocation in Chinese hamster ovary cells that concomitantly overexpressed human TLR4 and myeloid differentiation protein-2 (MD-2), but failed to modulate TLR4 or MD-2 cell surface expression. Pretreatment of 3E10/TLR2 cells with LAM or STF decreased their NF-kappaB responses induced by subsequent stimulation with these substances or LPS. Conversely, prior exposure of 3E10/TLR2 cells to LPS led to hyporesponsiveness to UPS, LAM, and STF, indicating that LPS and mycobacterial products induce cross-tolerance. Thus, tolerance to LPS and mycobacterial components cannot be attributed solely to a decrease in TLR/MD-2 expression levels, suggesting inhibition of expression or function of other signaling intermediates.