Identification of specific expressed genes of inflammatory Osteoclasts in joints of reheumatoid arthritis patients

目的：我们以前已经显示了在体外分化的单核细胞成破骨细胞样多核细胞的粒细胞-巨噬细胞集落刺激因子（GM-CSF），众所周知的破骨细胞生成的有效抑制剂。为探讨类风湿关节炎（RA）发病条件下破骨细胞样细胞的特性，我们将GM-CSF诱导的多核细胞与核因子κ B配体受体激活剂（RANKL）诱导的多核细胞进行比较。采用逆转录-聚合酶链反应（RT-PCR）检测基质金属蛋白酶（MMP）-12和6种破骨细胞标志物：抗酒石酸酸性磷酸酶（TRAP）、骨酸酐酶、组织蛋白酶K、降钙素受体、整合素αV和MMP-9的表达。结果：RA-SNCs共培养的单个核细胞表达破骨细胞标志物（MMP-9除外），并保持RANKL诱导的破骨细胞样细胞形成能力，RANKL诱导的细胞表达MMP-9及其他破骨细胞标志物，但不表达MMP-12。另一方面，通过GM-CSF的刺激，单个核细胞也分化为表达MMP-9和MMP-12的破骨细胞样多核细胞。结论：RA关节内存在两种不同类型的TRAP阳性多核细胞，其表达的MMP-12与RA关节内的TRAP阳性多核细胞表达的MMP-12呈负相关。GM-CSF诱导的细胞与RANKL诱导的细胞通过MMP-12的表达而区别，应该参与RA的病理过程。RA SNCs与滑膜巨噬细胞共培养是研究RA滑膜巨噬细胞分化机制及其在RA发病机制中的作用的一个非常有用的系统。

Objective : We have previously shown in vitro differentiation of monocytes into osteoclast-like multinucleated cells by granulocyte-macrophage colony-stimulating factor (GM-CSF), well-known as the potent inhibitor of osteoclastogenesis. We compared GM-CSF-induced multicleated cells with those induced by receptor activator of nuclear factor kappa B ligand (RANKL) to elucidate the attributes of osteoclast-like cells generated under the pathgenic condition of rheumatoid arthritis (RA).Methods : The precursors were produced by the co-culture of monocytes with synovial nurse cells derived fro RA joints (RA-SNCs) and then subjected to the formation of multinucleated cells. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to measure the expression of matrix metalloproteinase (MMP)-12 and six osteoclast markers ; tartrate-resistant acid phosphatase (TRAP), crbonic anhydrase, cathepsin K, calcitonin receptor, integirin αV and MMP-9. TRAP and MMP-12 in RA joints specimen were evaluated histochemically.Results : The mononuclear cells co-cultivated with RA-SNCs expressed ostoclast markers except for MMP-9, and maintained the ability of osteoclast-like cell formation induced by RANKL, RANKL-induced cells expressed MMP-9 as well as other ostoclast markers, but not MMP-12. On the other hand, by thestimulation of GM-CSF, the mononuclear cells also differentiated into osteoclast-like multinuleated cells expressing MMP-9 and MMP-12. Farthermore, the multinucleated cells producing MMP-12 were found in the erosive bone area of rheumatoid joints.Conclusion : These findings suggest the presence of two distinct types of TRAP-positive multinucleated cells in RA joints. GM-CSF-induced cells, distinguishable from RANKL-induced cells by the expression of MMP-12, should be involved in the pathology of RA. The co-culture with RA-SNCs is very useful system to investigate mechanism of the differentiation of synovial macrophages and their roles in the pathogenesis of RA.