The Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia.
Mol Immunol. 2010 Jul;47(11-12):2065-73. doi: 10.1016/j.molimm.2010.04.004. Epub 2010 May 6.
Production of chemokines in dendritic cells (DCs) may be crucial in modulating immune responses generated through Toll-like receptor (TLR)-mediated recognition of microbial products. We evaluated chemokine production in DCs induced by TLR agonists and investigated the role of signaling pathways. DCs were generated from mouse bone marrow cells cultured with Fms-like tyrosine kinase-3 ligand and stimulated with a wide array of individual TLR agonists or simultaneously with pairs of combinations. Production of monocyte chemoattractant protein-1 (MCP-1/CCL2), macrophage inflammatory protein-1 (MIP-1/CCL3) and regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), were determined in cell culture supernatants by ELISA or cytokine cytometric bead array. Pharmacological inhibitors of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), nuclear factor-kappaB (NF-kB) and phosphatidylinositol 3-kinase (PI3K), were used to investigate the role of signaling pathways. TLR agonists induced significantly elevated MCP-1, RANTES, and MIP-1. Production of RANTES and MIP-1 was particularly prominent after stimulation of DCs with TLR3 (Poly(I:C)), and TLR7/8 (R848) or TLR9 (CpG ODN) agonists, respectively. However, down-modulation of chemokine production was observed in simultaneously TLR-stimulated DCs. A positive role was identified for NF-kB, PI3K and ERK, whereas JNK had a negative regulatory effect on chemokine production in DCs. Positive and negative regulatory roles for the p38 MAPK pathway were observed. Thus, chemokine levels differed and most notably there was down-modulation of chemokines in DCs stimulated with combined TLR agonists. Furthermore, analysis of signaling pathways revealed a role for MAPKs in positive and negative regulation of chemokine production in DCs. The chemokine response of DCs induced by TLR agonists appears complex and could have important implications for vaccine design.
树突状细胞 (DCs) 中趋化因子的产生对于调节通过 Toll 样受体 (TLR) 介导的微生物产物识别而产生的免疫反应可能至关重要。我们评估了 TLR 激动剂诱导的 DC 中趋化因子的产生，并研究了信号通路的作用。使用 Fms 样酪氨酸激酶-3 配体从小鼠骨髓细胞中生成 DC，并使用广泛的单个 TLR 激动剂或同时使用对组合对其进行刺激。通过 ELISA 或细胞因子流式细胞术微珠阵列在细胞培养上清液中测定单核细胞趋化蛋白-1 (MCP-1/CCL2)、巨噬细胞炎性蛋白-1 (MIP-1/CCL3) 和调节激活正常 T 细胞表达和分泌 (RANTES/CCL5) 的产生。使用丝裂原活化蛋白激酶 (MAPK)、细胞外信号调节激酶 (ERK)、c-Jun N 末端激酶 (JNK)、核因子-κB (NF-κB) 和磷脂酰肌醇 3-激酶 (PI3K) 的药理学抑制剂来研究信号通路的作用。TLR 激动剂诱导显著升高的 MCP-1、RANTES 和 MIP-1。在用 TLR3（Poly(I:C)）、TLR7/8（R848）或 TLR9（CpG ODN）激动剂刺激 DC 后，RANTES 和 MIP-1 的产生尤为明显。然而，在同时 TLR 刺激的 DC 中观察到趋化因子产生的下调。鉴定出 NF-κB、PI3K 和 ERK 发挥了积极作用，而 JNK 对 DC 中趋化因子的产生具有负调节作用。p38 MAPK 途径表现出正调节和负调节作用。因此，趋化因子水平不同，在用联合 TLR 激动剂刺激的 DC 中，趋化因子明显下调。此外，信号通路分析表明 MAPK 在 DC 中趋化因子产生的正调节和负调节中发挥作用。TLR 激动剂诱导的 DC 趋化因子反应复杂，这可能对疫苗设计具有重要意义。
