类风湿关节炎滑膜成纤维细胞中模式识别受体核苷酸结合寡聚化结构域2的表达、调控及信号传导

Expression, regulation, and signaling of the pattern-recognition receptor nucleotide-binding oligomerization domain 2 in rheumatoid arthritis synovial fibroblasts.

作者信息

Ospelt Caroline, Brentano Fabia, Jüngel Astrid, Rengel Yvonne, Kolling Christoph, Michel Beat A, Gay Renate E, Gay Steffen

机构信息

Center of Experimental Rheumatology, University Hospital Zurich, and Zurich Center of Integrative Human Physiology, University of Zurich, Zurich, Switzerland.

出版信息

Arthritis Rheum. 2009 Feb;60(2):355-63. doi: 10.1002/art.24226.

DOI:10.1002/art.24226

PMID:19180502

Abstract

OBJECTIVE

Since pattern-recognition receptors (PRRs), in particular Toll-like receptors (TLRs), were found to be overexpressed in the synovium of rheumatoid arthritis (RA) patients and to play a role in the production of disease-relevant molecules, we sought to determine the expression, regulation, and function of the PRR nucleotide-binding oligomerization domain 2 (NOD-2) in RA.

METHODS

Expression of NOD-2 in synovial tissues was analyzed by immunohistochemistry. Expression and induction of NOD-2 in RA synovial fibroblasts (RASFs) were measured by conventional and real-time polymerase chain reaction (PCR) analyses. Levels of interleukin-6 (IL-6) and IL-8 were measured by enzyme-linked immunosorbent assay (ELISA) and expression of matrix metalloproteinases (MMPs) by ELISA and/or real-time PCR. NOD-2 expression was silenced with small interfering RNA. Western blotting with antibodies against phosphorylated and total p38, JNK, and ERK, as well as inhibitors of p38, JNK, and ERK was performed. Activation of NF-kappaB was measured by electrophoretic mobility shift assay.

RESULTS

NOD-2 was expressed by fibroblasts and macrophages in the synovium of RA patients, predominantly at sites of invasion into articular cartilage. In cultured RASFs, no basal expression of messenger RNA for NOD-2 was detectable, but was induced by poly(I-C), lipopolysaccharide, and tumor necrosis factor alpha. After up-regulation of NOD-2 by TLR ligands, its ligand muramyl dipeptide (MDP) increased the expression of IL-6 and IL-8 via p38 and NF-kappaB. Stimulation with MDP further induced the expression of MMP-1, MMP-3, and MMP-13.

CONCLUSION

Not only TLRs, but also the PRR NOD-2 is expressed in the synovium of RA patients, and activation of NOD-2 acts synergistically with TLRs in the production of proinflammatory and destructive mediators. Therefore, NOD-2 might contribute to the initiation and perpetuation of chronic, destructive inflammation in RA.

摘要

目的

由于模式识别受体（PRR），特别是 Toll 样受体（TLR），在类风湿关节炎（RA）患者的滑膜中过度表达，并在疾病相关分子的产生中发挥作用，我们试图确定 PRR 核苷酸结合寡聚化结构域 2（NOD-2）在 RA 中的表达、调控及功能。

方法

通过免疫组织化学分析滑膜组织中 NOD-2 的表达。通过传统及实时聚合酶链反应（PCR）分析测量 RA 滑膜成纤维细胞（RASF）中 NOD-2 的表达及诱导情况。通过酶联免疫吸附测定（ELISA）测量白细胞介素-6（IL-6）和 IL-8 的水平，并通过 ELISA 和/或实时 PCR 测量基质金属蛋白酶（MMP）的表达。用小干扰 RNA 使 NOD-2 表达沉默。用针对磷酸化和总 p38、JNK 和 ERK 的抗体以及 p38、JNK 和 ERK 的抑制剂进行蛋白质印迹分析。通过电泳迁移率变动分析测量核因子κB（NF-κB）的激活情况。

结果

NOD-2 在 RA 患者滑膜中的成纤维细胞和巨噬细胞中表达，主要在侵入关节软骨的部位。在培养的 RASF 中，未检测到 NOD-2 信使核糖核酸的基础表达，但可被聚肌苷酸-聚胞苷酸（poly(I-C)）、脂多糖和肿瘤坏死因子α诱导。在 TLR 配体上调 NOD-2 后，其配体胞壁酰二肽（MDP）通过 p38 和 NF-κB 增加 IL-6 和 IL-8 的表达。用 MDP 刺激进一步诱导 MMP-1、MMP-3 和 MMP-13 的表达。