Dai C H, Price J O, Brunner T, Krantz S B
Hematology Division, Department of Medicine, Department of Veterans Affairs Medical Center (DVAMC) and Vanderbilt University School of Medicine, Nashville, TN 37232-6305, USA.
Blood. 1998 Feb 15;91(4):1235-42.
Interferon gamma (IFNgamma) inhibits the growth and differentiation of highly purified human erythroid colony-forming cells (ECFCs) and induces erythroblast apoptosis. These effects are dose- and time-dependent. Because the cell surface receptor known as Fas (APO-1; CD95) triggers programmed cell death after activation by its ligand and because incubation of human ECFCs with IFNgamma produces apoptosis, we have investigated the expression and function of Fas and Fas ligand (FasL) in highly purified human ECFCs before and after incubation with IFNgamma in vitro. Only a small percentage of normal human ECFCs express Fas and this is present at a low level as detected by Northern blotting for the Fas mRNA and flow cytometric analysis of Fas protein using a specific mouse monoclonal antibody. The addition of IFNgamma markedly increased the percentage of cells expressing Fas on the surface of the ECFCs as well as the intensity of Fas expression. Fas mRNA was increased by 6 hours, whereas Fas antigen on the cell surface increased by 24 hours, with a plateau at 72 hours. This increase correlated with the inhibitory effect of IFNgamma on ECFC proliferation. CH-11 anti-Fas antibody, which mimics the action of the natural FasL, greatly enhanced IFNgamma-mediated suppression of cell growth and production of apoptosis, indicating that Fas is functional. Expression of FasL was also demonstrated in normal ECFCs by reverse transcriptase-polymerase chain reaction and flow cytometric analysis with specific monoclonal antibody. FasL was constitutively expressed among erythroid progenitors as they matured from day 5 to day 8 and IFNgamma treatment did not change this expression. Apoptosis induced by IFNgamma was greatly reduced by the NOK-2 antihuman FasL antibody and an engineered soluble FasL receptor, Fas-Fc, suggesting that Fas-FasL interactions among the ECFCs produce the erythroid inhibitory effects and apoptosis initiated by IFNgamma.
干扰素γ（IFNγ）可抑制高度纯化的人红系集落形成细胞（ECFCs）的生长和分化，并诱导成红细胞凋亡。这些效应具有剂量和时间依赖性。由于名为Fas（APO-1；CD95）的细胞表面受体在被其配体激活后会触发程序性细胞死亡，且人ECFCs与IFNγ孵育会产生凋亡，因此我们研究了体外与IFNγ孵育前后高度纯化的人ECFCs中Fas和Fas配体（FasL）的表达及功能情况。正常人类ECFCs中只有一小部分表达Fas，通过Northern印迹法检测Fas mRNA以及使用特异性小鼠单克隆抗体进行Fas蛋白的流式细胞术分析发现其表达水平较低。添加IFNγ显著增加了ECFCs表面表达Fas的细胞百分比以及Fas表达强度。Fas mRNA在6小时时增加，而细胞表面的Fas抗原在24小时时增加，72小时时达到平台期。这种增加与IFNγ对ECFC增殖的抑制作用相关。模拟天然FasL作用的CH-11抗Fas抗体极大地增强了IFNγ介导的细胞生长抑制和凋亡产生，表明Fas具有功能。通过逆转录-聚合酶链反应以及使用特异性单克隆抗体进行流式细胞术分析，也证实了正常ECFCs中FasL的表达。从第5天到第8天，随着红系祖细胞成熟，FasL呈组成性表达，IFNγ处理并未改变这种表达。NOK-2抗人FasL抗体和工程化可溶性FasL受体Fas-Fc可显著减少IFNγ诱导的凋亡，这表明ECFCs之间的Fas-FasL相互作用产生了IFNγ引发的红系抑制效应和凋亡。
