Kirino Yohei, Takeno Mitsuhiro, Murakami Shuji, Kobayashi Masayoshi, Kobayashi Hideo, Miura Kenji, Ideguchi Haruko, Ohno Shigeru, Ueda Atsuhisa, Ishigatsubo Yoshiaki
Yokohama City University, Graduate School of Medicine, Yokohama, Japan.
Arthritis Rheum. 2007 Feb;56(2):464-75. doi: 10.1002/art.22370.
To examine the interaction between heme oxygenase 1 (HO-1), a stress-induced antiinflammatory protein, and tumor necrosis factor alpha (TNFalpha) in human peripheral blood monocytes.
Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors or from patients with rheumatoid arthritis (RA) receiving the anti-tumor necrosis factor alpha (anti-TNFalpha) monoclonal antibody infliximab. CD14+ cells were isolated by magnetic cell sorting, cultured with TNFalpha or auranofin, and transfected with a plasmid encoding HO-1 or an HO-1-specific small interfering RNA vector. Protein and messenger RNA (mRNA) levels were examined by immunoblotting and real-time polymerase chain reaction. Cytokine levels in culture supernatants were measured by enzyme-linked immunosorbent assay. HO-1 gene transcription was evaluated using a luciferase reporter gene assay. Actinomycin D and cycloheximide were used to monitor the stability of mRNA and protein.
HO-1 is constitutively expressed by CD14+ PBMCs from healthy donors. TNFalpha suppressed HO-1 expression by accelerating the decay of mRNA without affecting gene transcription or protein stability. Forced expression or selective knock-down of the HO-1 gene expression resulted in down-regulation or up-regulation, respectively, of proinflammatory cytokine synthesis by monocytes. Treatment with infliximab significantly increased HO-1 mRNA levels and reduced TNFalpha synthesis by PBMCs from RA patients.
TNFalpha accelerated inflammatory responses by down-regulating HO-1 expression in human monocytes. TNF antagonists may block this TNF-dependent suppression of HO-1 expression, resulting in an amelioration of inflammation.
研究应激诱导的抗炎蛋白血红素加氧酶1（HO-1）与人类外周血单核细胞中肿瘤坏死因子α（TNFα）之间的相互作用。
从健康供体或接受抗肿瘤坏死因子α（抗TNFα）单克隆抗体英夫利昔单抗治疗的类风湿关节炎（RA）患者中获取外周血单个核细胞（PBMC）。通过磁珠细胞分选法分离CD14+细胞，用TNFα或金诺芬培养，并转染编码HO-1的质粒或HO-1特异性小干扰RNA载体。通过免疫印迹和实时聚合酶链反应检测蛋白质和信使核糖核酸（mRNA）水平。用酶联免疫吸附测定法测量培养上清液中的细胞因子水平。使用荧光素酶报告基因测定法评估HO-1基因转录。放线菌素D和环己酰亚胺用于监测mRNA和蛋白质的稳定性。
健康供体的CD14+ PBMC组成性表达HO-1。TNFα通过加速mRNA降解抑制HO-1表达，但不影响基因转录或蛋白质稳定性。HO-1基因表达的强制表达或选择性敲低分别导致单核细胞促炎细胞因子合成的下调或上调。英夫利昔单抗治疗显著增加RA患者PBMC的HO-1 mRNA水平并降低TNFα合成。
TNFα通过下调人类单核细胞中的HO-1表达加速炎症反应。TNF拮抗剂可能阻断这种TNF依赖性的HO-1表达抑制，从而改善炎症。
