Impaired mitogen-activated protein kinase activation and altered cytokine secretion in endotoxin-tolerant human monocytes
Impaired mitogen-activated protein kinase activation and altered cytokine secretion in endotoxin-tolerant human monocytes
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DOI：
10.1097/00005373-200011000-00003
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发表时间：
2000-11-01
影响因子：
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通讯作者：
West, MA
中科院分区：
文献类型：
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作者：
Heagy, W;Hansen, C;West, MA
背景：暴露于多种炎症刺激后单核细胞/巨噬细胞细胞因子产生失调可能导致多器官衰竭和败血症。内毒素（脂多糖[LPS]）激活小鼠巨噬细胞导致丝裂原活化蛋白激酶级联中激酶的磷酸化。LPS预处理小鼠巨噬细胞诱导LPS耐受，抑制LPS刺激的激酶(ERI（YZ和p38）的激活，减少肿瘤坏死因子（TNF）的释放。我们试图确定在LPS耐受的人外周血单核细胞中是否存在类似的LPS依赖性信号转导改变。取健康志愿捐献者的人外周血单核细胞（n = 12），在RPMI 1640 +/-10 ng/mL LPS培养基中培养18小时，然后用0 ~ 1000 ng/mL LPS刺激，5小时后用酶联免疫吸附法测定上清TNF和白细胞介素-l -l水平。p42/p44激酶（ERK1/2）的激活在LPS后15分钟，使用新的流式细胞术方法用双磷酸化（活性）ERK1/2的单克隆抗体检测。结果：LPS耐受（10 ng/mL LPS预处理）的人单核细胞对LPS刺激的TNF分泌有明显的抑制作用，但il - 1释放增强（P < 0.05）； LPS初始刺激后，非LPS耐受的人单核细胞erk1 /2阳性细胞比例显著增加，而LPS耐受的细胞没有出现这种情况。结论：人单核细胞的脂多糖耐受性与抑制脂多糖刺激的TNF分泌、il -1释放增加和丝裂原活化的蛋白激酶级联（ERK1/2）激活缺陷有关，这些结果提示了一种鉴定脓毒症患者脂多糖耐受性和单核细胞功能障碍的方法。
Background: Dysregulation of monocyte/macrophage cytokine production after exposure to multiple inflammatory stimuli may contribute to multiple organ failure and sepsis. Endotoxin (lipopolysaccharide [LPS]) activation of murine macrophage results in the phosphorylation of kinases in the mitogen-activated protein kinase cascade. Pretreatment of murine macrophages with LPS induces LPS-tolerance, with inhibition of LPS-stimulated activation of kinases (ERI(YZ and p38) and diminished release of tumor necrosis factor (TNF), We sought to determine whether similar alterations in LPS-dependent signal transduction are present in LPS-tolerant human peripheral blood monocytes,Methods: Human peripheral blood monocytes from healthy volunteer donors (n = 12) were incubated in RPMI 1640 culture medium +/-10 ng/mL of LPS for 18 hours, then stimulated with 0 to 1,000 ng/mL of LPS, Supernatant TNF and interleukin-l OL-l) levels were measured after 5 hours by enzyme-linked immunosorbent assay. Activation of the p42/p44 kinases (ERK1/2) was measured 15 minutes after LPS with monoclonal antibodies to diphosphorylated (active) ERK1/2 using novel flow cytometric methods.Results: LPS-tolerant (10 ng/mL LPS pretreatment) human monocytes had significant inhibition of LPS-stimulated TNF secretion but augmented IL-I release (P < 0.05), Nontolerant human monocytes had a dramatic increase in the percentage of ERK1/2-positive cells in response to an initial stimulation with LPS, This did not occur in the LPS-tolerant cells. Phorbol-12-myristate-13 acetate restored ERK1/2 activation in LPS-tolerant humanConclusion: LPS-tolerance in human monocytes is associated with inhibition of LPS-stimulated TNF secretion, augmented release of IL-l, and defective activation of mitogen-activated protein kinase cascade (ERK1/2), These results suggest a method of identifying LPS-tolerance and monocyte dysfunction in patients with sepsis.