内毒素耐受的人单核细胞中丝裂原活化蛋白激酶激活受损及细胞因子分泌改变。

Impaired mitogen-activated protein kinase activation and altered cytokine secretion in endotoxin-tolerant human monocytes.

作者信息

Heagy W, Hansen C, Nieman K, Rodriguez J L, West M A

机构信息

Department of Surgery, Hennepin County Medical Center, Minneapolis Medical Research Foundation, University of Minnesota 55415, USA.

出版信息

J Trauma. 2000 Nov;49(5):806-14. doi: 10.1097/00005373-200011000-00003.

DOI:10.1097/00005373-200011000-00003

PMID:11086768

Abstract

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 (ERK1/2 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-1 (IL-1) 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-1 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 human monocytes.

CONCLUSION

LPS-tolerance in human monocytes is associated with inhibition of LPS-stimulated TNF secretion, augmented release of IL-1, 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.

摘要

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功能衰竭和脓毒症。内毒素（脂多糖 [LPS]）激活小鼠巨噬细胞会导致丝裂原活化蛋白激酶级联反应中的激酶磷酸化。用 LPS 预处理小鼠巨噬细胞可诱导 LPS 耐受，抑制 LPS 刺激的激酶（ERK1/2 和 p38）激活，并减少肿瘤坏死因子（TNF）的释放。我们试图确定 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。

方法

将来自健康志愿者供体（n = 12）的人外周血单核细胞在含有或不含 10 ng/mL LPS 的 RPMI 1640 培养基中孵育 18 小时，然后用 0 至 1000 ng/mL 的 LPS 刺激。5 小时后通过酶联免疫吸附测定法测量上清液中 TNF 和白细胞介素-1（IL-1）的水平。使用新型流式细胞术方法，在 LPS 刺激 15 分钟后，用针对双磷酸化（活性）ERK1/2 的单克隆抗体测量 p42/p44 激酶（ERK1/2）的激活情况。

结果

LPS 耐受（10 ng/mL LPS 预处理）的人单核细胞对 LPS 刺激的 TNF 分泌有显著抑制，但 IL-1 释放增加（p < 0.05）。未耐受的人单核细胞在初次用 LPS 刺激后，ERK1/2 阳性细胞的百分比显著增加。而 LPS 耐受细胞中未出现这种情况。佛波醇-12-肉豆蔻酸酯-13-乙酸酯可恢复 LPS 耐受的人单核细胞中 ERK1/2 的激活。