Institute of Immunology, University of Oslo and Rikshospitalet University Hospital, Oslo, Norway.
Mol Immunol. 2010 Mar;47(6):1226-34. doi: 10.1016/j.molimm.2009.12.015. Epub 2010 Jan 22.
Meconium, the first intestinal discharge of the newborn, contains material accumulated during fetal life. Meconium activates complement and CD14 and may induce a systemic inflammatory response. Toll-like receptors are classical pattern-recognition receptors recognizing both exogenous and host-derived ligands. The cyanobacterial product CyP is a potent LPS antagonist binding to the TLR4/MD-2 complex. The aim of the present study was to investigate the role of the CD14/TLR4/MD-2 complex in meconium-induced inflammation.
Whole blood from six donors was preincubated with anti-CD14 or CyP. Meconium was added and the samples were incubated for 4h. Twenty-seven inflammatory mediators were measured in a Bioplex Array Reader. Human embryonic kidney cells transfected with plasmids containing NF-kappaB dependent luciferase reporter, human MD-2, TLR4, TLR2 and/or CD14, were incubated with meconium or LPS for 18 h. Luciferase activity in cytoplasmic extracts was measured using a Luciferase Assay System kit.
Meconium induced formation of a broad panel of inflammatory mediators. CyP and anti-CD14 significantly (p<0.001) inhibited meconium-induced formation of (a) proinflammatory cytokines (TNF-alpha, IL-1beta, IL-6, IFN-gamma) by 60-80% and 72-94%, respectively, (b) anti-inflammatory cytokines (IL-10, IL-1Ra) by 58-59% and 50-65%, respectively, (c) chemokines (IL-8, MCP-1, MIP-1alpha, MIP-1beta, eotaxin, IP-10) by 43-77% and 57-87%, respectively, and (d) growth factors (G-CSF, GM-CSF, basic FGF, PDGFbb) by 53-71% and 40-78%, respectively, with no statistical significant difference between Cyp and anti-CD14. The inflammatory response could only partly be explained by LPS, suggesting that endogenous components of meconium contribute to the inflammatory response. Meconium activated NF-kappaB dose-dependently in cells expressing TLR4/MD-2 together with CD14, while no effect was seen in cells expressing TLR4/MD-2 alone or in TLR2/CD14 transfected cells.
The results indicate that the CD14-dependent meconium-induced inflammatory reaction is mediated through the TLR4/MD2 complex. These data may have implications for future therapeutic strategies for meconium aspiration syndrome.
胎粪是新生儿的第一次肠道排泄物，其中包含胎儿期积累的物质。胎粪激活补体和 CD14，并可能诱导全身炎症反应。Toll 样受体是识别外源性和宿主来源配体的经典模式识别受体。蓝藻产物 CyP 是一种有效的 LPS 拮抗剂，与 TLR4/MD-2 复合物结合。本研究旨在探讨 CD14/TLR4/MD-2 复合物在胎粪诱导炎症中的作用。
六位供体的全血先用抗 CD14 或 CyP 预孵育。加入胎粪并孵育 4 小时。在 Bioplex 阵列读取器中测量 27 种炎症介质。转染含 NF-κB 依赖性荧光素酶报告基因、人 MD-2、TLR4、TLR2 和/或 CD14 的人胚肾细胞与胎粪或 LPS 孵育 18 小时。使用荧光素酶测定试剂盒测量细胞质提取物中的荧光素酶活性。
胎粪诱导形成广泛的炎症介质谱。CyP 和抗 CD14 显著（p<0.001）抑制胎粪诱导形成的（a）促炎细胞因子（TNF-α、IL-1β、IL-6、IFN-γ），分别为 60-80%和 72-94%，（b）抗炎细胞因子（IL-10、IL-1Ra），分别为 58-59%和 50-65%，（c）趋化因子（IL-8、MCP-1、MIP-1α、MIP-1β、eotaxin、IP-10），分别为 43-77%和 57-87%，（d）生长因子（G-CSF、GM-CSF、碱性 FGF、PDGFbb），分别为 53-71%和 40-78%，CyP 和抗 CD14 之间无统计学显著差异。炎症反应只能部分由 LPS 解释，表明胎粪中的内源性成分有助于炎症反应。胎粪在表达 TLR4/MD-2 以及 CD14 的细胞中呈剂量依赖性激活 NF-κB，而在仅表达 TLR4/MD-2 的细胞或转染 TLR2/CD14 的细胞中则无此作用。
结果表明，CD14 依赖性胎粪诱导的炎症反应是通过 TLR4/MD2 复合物介导的。这些数据可能对胎粪吸入综合征的未来治疗策略具有重要意义。
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