Bobacz K, Sunk I G, Hofstaetter J G, Amoyo L, Toma C D, Akira S, Weichhart T, Saemann M, Smolen J S
Medical University of Vienna, Vienna, Austria.
Arthritis Rheum. 2007 Jun;56(6):1880-93. doi: 10.1002/art.22637.
To assess the presence of Toll-like receptors (TLRs) 1-9 in human articular cartilage, and to investigate the effects of lipopolysaccharide (LPS)-induced activation of TLR-4 on biosynthetic activity and matrix production by human articular chondrocytes.
TLRs 1-9 were assessed in human articular cartilage by reverse transcription-polymerase chain reaction (RT-PCR); TLR-4 was also analyzed by Western blotting and immunohistochemistry. Articular chondrocytes were isolated from human donors and from wild-type or TLR-4(-/-) mice. Chondrocyte monolayer cultures were incubated with interleukin-1beta (IL-1beta) and LPS in the absence or presence of bone morphogenetic protein 7 (BMP-7) and IL-1 receptor antagonist (IL-1Ra). Neosynthesis of sulfated glycosaminoglycans (sGAG) was measured by (35)S-sulfate incorporation. Endogenous gene expression of cartilage markers as well as IL-1beta was examined using RT-PCR. The involvement of p38 kinase or p44/42 kinase (ERK-1/2) in LPS-mediated TLR-4 signaling was investigated by immunoblotting, RT-PCR, and sGAG synthesis.
TLRs 1-9 were found on the messenger RNA (mRNA) level in human articular chondrocytes. The presence of TLR-4 was also observed on the protein level. In murine and human articular chondrocytes, but not in chondrocytes derived from TLR-4(-/-) mice, stimulation with LPS resulted in a decrease in total proteoglycan synthesis. IL-1beta mRNA expression was increased by TLR-4 activation, whereas expression of aggrecan and type II collagen was significantly decreased. The presence of BMP-7 and IL-1Ra antagonized the anti-anabolic effects of LPS. Blocking of p38, but not ERK-1/2, resulted in inhibition of both LPS-mediated IL-1beta gene expression and the negative effects of LPS on matrix biosynthesis.
These data demonstrate the presence of TLRs in human articular cartilage. The suppressive effects of LPS on cartilage biosynthetic activity are dependent on the presence of TLR-4, are governed, at least in part, by an up-regulation of IL-1beta, and are mediated by p38 kinase. These in vitro data indicate an anti-anabolic effect of TLR-4 in articular chondrocytes that may hamper cartilage repair in various joint diseases.
评估Toll样受体（TLRs）1 - 9在人关节软骨中的存在情况，并研究脂多糖（LPS）诱导的TLR - 4激活对人关节软骨细胞生物合成活性和基质产生的影响。
通过逆转录 - 聚合酶链反应（RT - PCR）评估人关节软骨中TLRs 1 - 9；还通过蛋白质印迹法和免疫组织化学分析TLR - 4。从人供体以及野生型或TLR - 4基因敲除（- / -）小鼠中分离关节软骨细胞。软骨细胞单层培养物在不存在或存在骨形态发生蛋白7（BMP - 7）和白细胞介素 - 1受体拮抗剂（IL - 1Ra）的情况下与白细胞介素 - 1β（IL - 1β）和LPS一起孵育。通过（35）S - 硫酸盐掺入法测量硫酸化糖胺聚糖（sGAG）的新合成。使用RT - PCR检测软骨标志物以及IL - 1β的内源性基因表达。通过免疫印迹、RT - PCR和sGAG合成研究p38激酶或p44/42激酶（ERK - 1/2）在LPS介导的TLR - 4信号传导中的作用。
在人关节软骨细胞的信使核糖核酸（mRNA）水平上发现了TLRs 1 - 9。在蛋白质水平上也观察到了TLR - 4的存在。在小鼠和人关节软骨细胞中，但在源自TLR - 4（- / -）小鼠的软骨细胞中未观察到，LPS刺激导致总蛋白聚糖合成减少。TLR - 4激活增加了IL - 1βmRNA表达，而聚集蛋白聚糖和II型胶原的表达显著降低。BMP - 7和IL - 1Ra的存在拮抗了LPS的抗合成代谢作用。阻断p38而非ERK - 1/2导致抑制LPS介导的IL - 1β基因表达以及LPS对基质生物合成的负面影响。
这些数据证明了TLRs在人关节软骨中的存在。LPS对软骨生物合成活性的抑制作用依赖于TLR - 4的存在，至少部分受IL - 1β上调的控制，并由p38激酶介导。这些体外数据表明TLR - 4在关节软骨细胞中具有抗合成代谢作用，这可能会阻碍各种关节疾病中的软骨修复。
