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

目的：粒细胞-巨噬细胞集落刺激因子(GM-CSF)是破骨细胞生成的有效抑制因子，可在体外诱导单核细胞分化为破骨细胞样多核细胞。我们比较了GM-CSF诱导的多核细胞和核因子kappa B受体激活剂(RANKL)诱导的多核细胞，以阐明类风湿关节炎(RA)发病条件下产生的破骨细胞样细胞的特性。方法：通过单核细胞与RA关节滑膜滋养细胞(RA-SNCs)共同培养产生前体细胞，然后进行多核细胞的形成。逆转录-聚合酶链式反应检测基质金属蛋白酶-12和6种破骨细胞标志物：抗酒石酸酸性磷酸酶(TRAP)、强酸脱水酶、组织蛋白酶K、降钙素受体、整合素αV和基质金属蛋白酶-9的表达。结果：与RA-SNCs共培养的单个核细胞表达破骨细胞标志物，但不表达破骨细胞标志物，并维持RANKL诱导的破骨细胞样细胞的形成能力，RANKL诱导的细胞表达破骨细胞标志物，但不表达破骨细胞标志物。另一方面，经GM-CSF检测，单核细胞也分化为破骨细胞样多核细胞，表达基质金属蛋白酶-9和基质金属蛋白酶-12。结论：类风湿关节中存在两种不同类型的TRAP阳性多核细胞。GM-CSF诱导的细胞与RANKL诱导的细胞不同，其表达的基质金属蛋白酶-12可能参与了RA的病理过程。与RA-SNCs共培养是研究滑膜巨噬细胞分化机制及其在RA发病机制中作用的有效方法。

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.