CCL28-CCR10通路在类风湿关节炎单核细胞迁移中的作用

Role of the CCL28-CCR10 pathway in monocyte migration in rheumatoid arthritis

目的:观察类风湿关节炎(rheumatoid arthritis,RA)患者关节中单核/巨噬细胞趋化因子受体CCR10的表达,探讨趋化因子CCL28与其受体CCR10在RA单核细胞迁移中的作用及机制。方法:采用免疫组织化学法分析8例RA患者、4例骨关节炎(osteoarthritis,OA)患者和4例正常对照者滑膜组织中CCR10的表达并进行细胞染色评分(0~5分),流式细胞术检测26例RA患者和20例健康对照者外周血、15例RA患者滑液CD14^(+)单核细胞中CCR10阳性细胞比例,Transwell迁移实验检测CCL28对RA和健康对照单核细胞的趋化性,Western blotting检测CCL28干预RA单核细胞的细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)、蛋白激酶B(protein kinase B,Akt)通路磷酸化。结果:CCR10表达在RA滑膜衬里层细胞及衬里下层的巨噬细胞、血管内皮细胞、淋巴细胞;RA滑膜衬里层细胞和衬里下层巨噬细胞的CCR10表达明显高于OA和正常对照的滑膜(P均<0.01)。RA患者外周血CD14^(+)单核细胞表达CCR10明显高于健康对照者[(15.6±3.0)%vs.(7.7±3.8)%,P<0.01];RA患者滑液单核细胞CCR10的表达为(32.0±15.0)%,明显高于RA外周血(P<0.01)。体外实验中,10~100μg/L的CCL28能有效诱导RA和健康对照外周血CD14^(+)单核细胞迁移(P均<0.01);抗CCR10单抗能明显抑制CCL28对RA单核细胞的趋化(P<0.01)。CCL28干预RA单核细胞明显增加ERK和Akt的磷酸化(P均<0.05);ERK抑制剂(U0126)、磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)抑制剂(LY294002)可明显降低CCL28诱导的RA单核细胞迁移(P均<0.01)。结论:RA患者外周血、滑液及滑膜单核/巨噬细胞CCR10表达增高,CCL28与CCR10结合并通过激活ERK、PI3K/Akt信号通路促使RA单核细胞迁移;CCL28-CCR10通路可能参与招募单核细胞进入RA关节,从而促进滑膜炎症和骨破坏。

Objective:To examine the expression of chemokine receptor CCR10 on monocytes/macrophages in the joints of patients with rheumatoid arthritis(RA),and to investigate the role of chemokine CCL28 and its receptor CCR10 in the migration of RA monocytes and its mechanism.Methods:The expression of CCR10 in synovial tissues from 8 RA patients,4 osteoarthritis(OA)patients,and 4 normal controls was analyzed by immunohistochemistry,and cell staining was scored on a 0-5 scales.Flow cytometry was used to measure the percentage of CCR10 positive cells in CD14^(+)monocytes from peripheral blood of 26 RA patients and 20 healthy controls,as well as from synovial fluid of 15 RA patients.The chemotactic migration of monocytes from RA patients and healthy controls in response to CCL28 was evaluated using an in vitro Transwell system.Western blotting was conducted to assess phosphorylation of the extracellular signal-regulated kinase(ERK)and protein kinase B(Akt)pathways in RA monocytes upon CCL28 treatment.Results:CCR10 was predominantly expressed in RA synovial lining cells and sublining macrophages,endothelial cells,and lymphocytes.CCR10 expression was significantly increased on lining cells and sublining macrophages in RA synovial tissue compared with OA and normal synovial tissue(both P<0.01).The patients with RA had markedly elevated expression of CCR10 on peripheral blood CD14^(+)monocytes compared with the healthy controls[(15.6±3.0)%vs.(7.7±3.8)%,P<0.01].CCR10 expression on synovial fluid monocytes from the RA patients was(32.0±15.0)%,which was significantly higher than that on RA peripheral blood monocytes(P<0.01).In vitro,CCL28 caused significant migration of CD14^(+)monocytes from peripheral blood of the RA patients and the healthy controls at concentrations ranging from 10-100μg/L(all P<0.01).The presence of neutralizing antibody to CCR10 greatly suppressed CCL28-driven chemotaxis of RA monocytes(P<0.01).Stimulation of RA monocytes with CCL28 induced a remarkable increase in phosphorylation of ERK and Akt(both P<0.05).ERK inhibitor(U0126)and phosphatidylinositol 3-kinase(PI3K)inhibitor(LY294002)strongly reduced the migration of RA monocytes in response to CCL28(both P<0.01).Conclusion:RA patients had increased CCR10 expression on peripheral blood,synovial fluid,and synovial tissue monocytes/macrophages.CCL28 ligation to CCR10 promoted RA monocyte migration through activation of the ERK and PI3K/Akt signaling pathways.The CCL28-CCR10 pathway could participate in monocyte recruitment into RA joints,thereby contributing to synovial inflammation and bone destruction.