Extracellular heat shock protein-70 induces endotoxin tolerance in THP-1 cells
Extracellular heat shock protein-70 induces endotoxin tolerance in THP-1 cells
复制标题
DOI：
10.4049/jimmunol.177.10.7184
复制
发表时间：
2006-11-15
影响因子：
4.4
通讯作者：
Wong, Hector R.
中科院分区：
文献类型：
--
作者：
Aneja, Rajesh;Odoms, Kelli;Wong, Hector R.
最近的研究表明，热休克蛋白-70(HSP-70)是一种细胞内蛋白，存在于细胞外，并通过CD14/TLR4途径传递信号。在这项研究中，我们验证了细胞外HSP-70诱导内毒素(LPS)耐受的假设。用人单核细胞系(THP-1)进行初始剂量-反应实验，以确定不诱导核因子-kappaB活性的HSP-70亚阈值浓度。用亚阈值浓度(0.03mU g/mlHSP-70)预处理18h，再用内毒素(1mU g/ml)刺激4h，HSP-70可降低随后由内毒素介导的核因子-kappaB依赖的启动子活性，并伴随着上清液中肿瘤坏死因子水平的显著降低。此外，从人类志愿者分离的单核细胞，随后用HSP70预处理，表现出对内毒素的耐受，这是从取消的培养上清液中的肿瘤坏死因子水平所证明的。通过在100℃下煮沸HSP-70 1小时或用与HSP-70制剂中存在的同等浓度的内毒素进行预处理，进行了额外的实验以排除HSP-70受到内毒素污染的可能性。这些实验表明，耐受性的诱导并不次于内毒素污染。抗HSP-70抗体的中和实验证实了HSP-70诱导耐受的特异性。HSP-70可抑制脂多糖刺激后抑制因子kappa B-α的胞浆降解，抑制抑制因子kappa B的激活。热休克蛋白70预适应可降低脂多糖刺激后核因子-kappaB p65亚单位的磷酸化水平。这些数据表明，细胞外HSP-70在改变单个核细胞对随后的内毒素攻击的反应中扮演了一个新的角色。
Recent data suggest that heat shock protein-70 (HSP-70), an intracellular protein, can exist in the extracellular compartment and signal through the CD14/TLR4 pathway. In this study, we tested the hypothesis that extracellular HSP-70 induces endotoxin (LPS) tolerance. Using human monocyte cell line (THP-1), initial dose-response experiments were conducted to determine a subthreshold concentration of HSP-70 that does not induce NF-kappa B activity. Differentiated THP-1 cells were preconditioned with subthreshold concentration (0.03 mu g/ml HSP-70) for 18 h, followed by LPS stimulation (1 mu g/ml) for 4 h. Preconditioning with HSP-70 decreased subsequent LPS-mediated NF-kappa B-dependent promoter activity and was accompanied by significant decreases of supernatant TNF levels. Furthermore, human monocytes isolated from human volunteers, subsequently preconditioned with HSP70, demonstrated LPS tolerance as evidenced by abrogated supernatant TNF levels. Additional experiments were conducted to exclude the possibility of endotoxin contamination of HSP-70 by boiling HSP-70 at 100 degrees C for 1 h or preconditioning with equivalent concentrations of endotoxin as present in the HSP-70 preparation. These experiments indicated that induction of tolerance was not secondary to endotoxin contamination. Neutralization experiments with an anti-HSP-70 Ab confirmed the specificity of HSP-70 in tolerance induction. Preconditioning with HSP-70 attenuated cytosolic degradation of inhibitor kappa B-alpha and inhibited activation of inhibitor kappa B kinase following LPS stimulation. HSP-70 preconditioning decreased phosphorylation of the p65 subunit of NF-kappa B following LPS stimulation. These data suggest a novel role for extracellular HSP-70 in modifying mononuclear cell responses to subsequent LPS challenge.