Xiao M, Li Z H, McMahel J, Broxmeyer H E, Lu L
Department of Medicine (Hematology/Oncology), Indiana University School of Medicine, Indianapolis, USA.
J Hematother. 1996 Apr;5(2):171-7. doi: 10.1089/scd.1.1996.5.171.
Interleukin 12 (IL-12), a heterodimeric cytokine with potent biologic activity, was evaluated for effects on retroviral-mediated gene transduction into human myeloid progenitor cells in vitro. Cord blood CD34 cells were prestimulated with Steel factor (SLF), IL-3, GM-CSF, and erythropoietin (Epo) in the presence and absence of 5-80 ng/ml IL-12 for 40 hr in suspension culture prior to gene transduction using viral supernatant collected from a packaging cell line containing the pLNL6 vector encoding Neo sequences. After gene transduction, cells were assayed for colony formation stimulated by Epo, GM-CSF, IL-3, and SLF, and gene transduction efficiency was determined by the percentage of G418 resistant (R) colonies and confirmed by PCR analysis. IL-12 dose-dependently inhibited retroviral-mediated gene transduction into human cord blood CD34 granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitors. These suppressive effects could be neutralized by incubation of IL-12 with polyclonal antihuman IL-12. IL-12 had no inhibitory effects directly on colony formation. To understand the possible mechanisms for this suppression, ELISA assays were used to detect the release of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, which could potentially have been induced by IL-12 from CD34 cells. TNF-alpha protein release was significantly increased in CD34 cells incubated with IL-12. No detectable levels of IFN-gamma were noted. Anti-TNF-alpha, but not anti-IFN-gamma, blocked the inhibitory effects of IL-12 on gene transduction. Moreover, TNF-alpha, but not IFN-gamma, suppressed gene transfer to the same degree as IL-12. No change of amphotropic receptor mRNA expression was noted by Northern blot analysis in cells treated with or without IL-12. The results suggest that the suppressive effects of IL-12 on retroviral gene transduction are, at least in part, mediated by IL-12 induction of the release of TNF-alpha.
白细胞介素12（IL-12）是一种具有强大生物活性的异二聚体细胞因子，我们对其在体外对逆转录病毒介导的基因转导进入人髓系祖细胞的影响进行了评估。在使用从含有编码新霉素序列的pLNL6载体的包装细胞系收集的病毒上清液进行基因转导之前，将脐血CD34细胞在有无5-80 ng/ml IL-12的情况下，与 Steel因子（SLF）、IL-3、GM-CSF和促红细胞生成素（Epo）一起在悬浮培养中预刺激40小时。基因转导后，检测细胞在Epo、GM-CSF、IL-3和SLF刺激下的集落形成情况，并通过G418抗性（R）集落的百分比确定基因转导效率，并通过PCR分析进行确认。IL-12剂量依赖性地抑制逆转录病毒介导的基因转导进入人脐血CD34粒细胞-巨噬细胞（CFU-GM）和红系（BFU-E）祖细胞。这些抑制作用可以通过将IL-12与多克隆抗人IL-12孵育来中和。IL-12对集落形成没有直接抑制作用。为了了解这种抑制的可能机制，使用ELISA测定法检测干扰素（IFN）-γ和肿瘤坏死因子（TNF）-α的释放，它们可能由IL-12从CD34细胞中诱导产生。在与IL-12孵育的CD34细胞中，TNF-α蛋白释放显著增加。未检测到可检测水平的IFN-γ。抗TNF-α而非抗IFN-γ阻断了IL-12对基因转导的抑制作用。此外，TNF-α而非IFN-γ与IL-12对基因转移的抑制程度相同。通过Northern印迹分析，在用或不用IL-12处理的细胞中，未观察到双嗜性受体mRNA表达的变化。结果表明，IL-12对逆转录病毒基因转导的抑制作用至少部分是由IL-12诱导TNF-α释放介导的。
