Klawitter Marina, Hakozaki Michiyuki, Kobayashi Hiroshi, Krupkova Olga, Quero Lilian, Ospelt Caroline, Gay Steffen, Hausmann Oliver, Liebscher Thomas, Meier Ullrich, Sekiguchi Miho, Konno Shin-ichi, Boos Norbert, Ferguson Stephen J, Wuertz Karin
Competence Center for Applied Biotechnology and Molecular Medicine, University of Zurich, Zurich, Switzerland,
Eur Spine J. 2014 Sep;23(9):1878-91. doi: 10.1007/s00586-014-3442-4. Epub 2014 Jul 5.
Although inflammatory processes play an essential role in painful intervertebral disc (IVD) degeneration, the underlying regulatory mechanisms are not well understood. This study was designed to investigate the expression, regulation and importance of specific toll-like receptors (TLRs)--which have been shown to play an essential role e.g. in osteoarthritis--during degenerative disc disease.
The expression of TLRs in human IVDs was measured in isolated cells as well as in normal or degenerated IVD tissue. The role of IL-1β or TNF-α in regulating TLRs (expression/activation) as well as in regulating activity of down-stream pathways (NF-κB) and expression of inflammation-related genes (IL-6, IL-8, HSP60, HSP70, HMGB1) was analyzed.
Expression of TLR1/2/3/4/5/6/9/10 was detected in isolated human IVD cells, with TLR1/2/4/6 being dependent on the degree of IVD degeneration. Stimulation with IL-1β or TNF-α moderately increased TLR1/TLR4 mRNA expression (TNF-α only), and strongly increased TLR2 mRNA expression (IL-1β/TNF-α), with the latter being confirmed on the protein level. Stimulation with IL-1β, TNF-α or Pam3CSK4 (a TLR2-ligand) stimulated IL-6 and IL-8, which was inhibited by a TLR2 neutralizing antibody for Pam3CSK4; IL-1β and TNF-α caused NF-κB activation. HSP60, HSP70 and HMGB1 did not increase IL-6 or IL-8 and were not regulated by IL-1β/TNF-α.
We provide evidence that several TLRs are expressed in human IVD cells, with TLR2 possibly playing the most crucial role. As TLRs mediate catabolic and inflammatory processes, increased levels of TLRs may lead to aggravated disc degeneration, chronic inflammation and pain development. Especially with the identification of more endogenous TLR ligands, targeting these receptors may hold therapeutic promise.
尽管炎症过程在疼痛性椎间盘退变中起重要作用，但其潜在的调控机制尚未完全明确。本研究旨在探讨特定的Toll样受体（TLRs）——已证明其在骨关节炎等疾病中起重要作用——在椎间盘退变疾病中的表达、调控及重要性。
在分离的细胞以及正常或退变的椎间盘组织中检测TLRs在人椎间盘内的表达。分析白细胞介素-1β（IL-1β）或肿瘤坏死因子-α（TNF-α）在调节TLRs（表达/激活）以及调节下游通路（核因子κB，NF-κB）活性和炎症相关基因（IL-6、IL-8、热休克蛋白60，HSP60、热休克蛋白70，HSP70、高迁移率族蛋白B1，HMGB1）表达中的作用。
在分离的人椎间盘细胞中检测到TLR1/2/3/4/5/6/9/10的表达，其中TLR1/2/4/6的表达依赖于椎间盘退变程度。用IL-1β或TNF-α刺激适度增加了TLR1/TLR4 mRNA表达（仅TNF-α），并强烈增加了TLR2 mRNA表达（IL-1β/TNF-α），后者在蛋白水平得到证实。用IL-1β、TNF-α或Pam3CSK4（一种TLR2配体）刺激可刺激IL-6和IL-8，Pam3CSK4的TLR2中和抗体可抑制这种刺激；IL-1β和TNF-α导致NF-κB激活。HSP60、HSP70和HMGB1不会增加IL-6或IL-8，也不受IL-1β/TNF-α调节。
我们提供的证据表明，几种TLRs在人椎间盘细胞中表达，其中TLR2可能发挥最关键的作用。由于TLRs介导分解代谢和炎症过程，TLRs水平升高可能导致椎间盘退变加重、慢性炎症和疼痛发展。特别是随着更多内源性TLR配体的发现，靶向这些受体可能具有治疗前景。
