A whole blood assay to assess peripheral blood dendritic cell function in response to Toll-like receptor stimulation
A whole blood assay to assess peripheral blood dendritic cell function in response to Toll-like receptor stimulation
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DOI：
10.1016/j.jim.2005.12.008
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发表时间：
2006-03-20
影响因子：
2.2
通讯作者：
Haslett, PAJ
中科院分区：
文献类型：
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作者：
Ida, JA;Shrestha, N;Haslett, PAJ
我们报告了一种评估全血（WB）中外周血树突状细胞（DC）成熟和功能的实用技术，该技术需要最少的血容量并最大限度地减少临床标本的离体操作。我们确定了对髓系 (mDC) 和浆细胞 (pDC) 谱系中 DC 成熟标志物（包括 CCR7、CD25、CD80 和 CD83）以及细胞内细胞因子、肿瘤坏死因子-α (TNF-α) 和干扰素-α (IFN-α) 进行流式细胞术分析的最佳条件。我们证明了在用 Toll 样受体 (TLR) 2/1、3、4、7、8 和 9 的配体进行短期刺激后，DC 会产生浓度依赖性的这些细胞因子。动力学研究表明，在用某些 TLR 配体刺激后 2 至 3 小时，TNF-α 和 IFN-α 蛋白表达达到最大。最后，利用来自 8 名健康捐赠者的细胞，我们比较了 WB、新鲜分离的外周血单核细胞 (PBMC) 和冷冻保存的 PBMC 中 DC 对 TLR 激活的反应。我们发现，在冷冻保存后，TNF-α 反应基本上得以保留，但 IFN-α 反应大大减弱或完全消除。总之，我们认为外周 DC 功能的 WB 分析是一种快速、可靠且简单的评估临床标本中 TLR 功能的方法，从而避免了容易出现伪影的细胞纯化。冷冻保存对某些 DC 反应的主要影响进一步强化了使用新鲜血液的快速方法的理由。 (c) 2005 Elsevier B.V. 保留所有权利。
We report a practical technique to assess peripheral blood dendritic cell (DC) maturation and function in whole blood (WB), which requires minimal blood volumes and minimizes ex vivo manipulations of clinical specimens. We determined optimal conditions for flow cytometric analysis of markers of DC maturation, including CCR7, CD25, CD80 and CD83, and of the intracellular cytokines, tumor necrosis factor-alpha (TNF-alpha) and interferon-alpha (IFN-alpha), in both myeloid (mDC) and plasmacytoid (pDC) lineages. We demonstrate concentration-dependent production of these cytokines by DC following short-term stimulation with ligands to Toll-like receptors (TLRs) 2/1, 3, 4, 7, 8 and 9. Kinetic studies revealed maximal TNF-alpha and IFN-alpha protein expression at 2 to 3 h after stimulation with certain TLR ligands. Finally, utilizing cells from a cohort of eight healthy donors, we compared DC responses to TLR activation in WB, freshly isolated peripheral blood mononuclear cells (PBMC) and cryopreserved PBMC. We found that TNF-alpha responses were essentially preserved, but IFN-alpha responses were profoundly diminished or entirely abrogated following cryopreservation. In conclusion, we propose that WB analysis of peripheral DC function is a rapid, reliable and simple method to evaluate TLR function in clinical specimens, which obviates artifact-prone cell purification. The major impact of cryopreservation on some DC responses further strengthens the case for a rapid method that uses fresh blood. (c) 2005 Elsevier B.V. All rights reserved.