Uehori Junji, Matsumoto Misako, Tsuji Shoutaro, Akazawa Takashi, Takeuchi Osamu, Akira Shizuo, Kawata Tsutomu, Azuma Ichiro, Toyoshima Kumao, Seya Tsukasa
Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Osaka 537-8511, Japan.
Infect Immun. 2003 Aug;71(8):4238-49. doi: 10.1128/IAI.71.8.4238-4249.2003.
The Mycobacterium bovis bacillus Calmette-Guérin (BCG) cell wall skeleton (CWS) consists of mycolic acids, arabinogalactan, and peptidoglycan (PGN) and activates Toll-like receptor 2 (TLR2) and TLR4. Here we investigated the ability of the essential portion of highly purified BCG CWS to support the TLR agonist function by using the following criteria: myeloid dendritic cell (DC) maturation, i.e., tumor necrosis factor alpha (TNF-alpha) production and CD83/CD86 up-regulation. The purified PGN region was sufficient to activate TLR2 and TLR4 in mouse DCs and macrophages; in TLR2 and TLR4 double-knockout cells the BCG PGN-mediated TNF-alpha production ability was completely impaired. Likewise, stimulation with BCG CWS of HEK293 cells expressing either human TLR2 or TLR4, MD-2, and CD14 resulted in NF-kappa B activation as determined by a reporter assay. Notably, specific blockers of extracellular human TLR2 (an original cocktail of monoclonal antibodies TLR2.45 and TH2.1) and TLR4 (E5531) inhibited BCG CWS-mediated NF-kappa B activation by 80%. Using this human TLR blocking system, we tested whether human myeloid DC maturation was TLR2 and TLR4 dependent. BCG PGN-mediated DC maturation was blocked by 70% by suppression of both TLR2 and TLR4 and by 30 to 40% by suppression of either of these TLRs. Similar but less profound suppression of BCG CWS-mediated DC maturation was observed. Hence, the presence of BCG PGN is a minimal requirement for activation of both TLR2 and TLR4 in human DCs, unlike the presence of PGNs of gram-positive bacteria, which activate only TLR2. Unexpectedly, however, BCG PGN, unlike BCG CWS, barely activated NF-kappa B in HEK293 cells coexpressing TLR2 plus TLR1, TLR2 plus TLR4, TLR2 plus TLR6, or TLR2 plus TLR10, suggesting that PGN receptors other than TLR2 and TLR4 present on human DCs but not on HEK293 cells are involved in TLR signaling for DC activation.
牛分枝杆菌卡介苗（BCG）细胞壁骨架（CWS）由分枝菌酸、阿拉伯半乳聚糖和肽聚糖（PGN）组成，可激活Toll样受体2（TLR2）和TLR4。在此，我们使用以下标准研究了高度纯化的BCG CWS的关键部分支持TLR激动剂功能的能力：髓样树突状细胞（DC）成熟，即肿瘤坏死因子α（TNF-α）的产生和CD83/CD86的上调。纯化的PGN区域足以激活小鼠DC和巨噬细胞中的TLR2和TLR4；在TLR2和TLR4双敲除细胞中，BCG PGN介导的TNF-α产生能力完全受损。同样，用BCG CWS刺激表达人TLR2或TLR4、MD-2和CD14的HEK293细胞，通过报告基因检测确定导致NF-κB激活。值得注意的是，细胞外人类TLR2（单克隆抗体TLR2.45和TH2.1的原始混合物）和TLR4（E5531）的特异性阻滞剂可将BCG CWS介导的NF-κB激活抑制80%。使用这种人类TLR阻断系统，我们测试了人类髓样DC成熟是否依赖于TLR2和TLR4。通过抑制TLR2和TLR4，BCG PGN介导的DC成熟被阻断70%，通过抑制这两种TLR中的任何一种，被阻断30%至40%。观察到BCG CWS介导的DC成熟有类似但不太明显的抑制。因此，与仅激活TLR2的革兰氏阳性菌PGN不同，BCG PGN的存在是激活人类DC中TLR2和TLR4的最低要求。然而，出乎意料的是，与BCG CWS不同，BCG PGN在共表达TLR2加TLR1、TLR2加TLR4、TLR2加TLR6或TLR2加TLR10的HEK293细胞中几乎不激活NF-κB，这表明人类DC上存在但HEK293细胞上不存在的除TLR2和TLR4之外的PGN受体参与了DC激活的TLR信号传导。
