Mechanisms of suppression on cytokine expression in HTLV-1-infected T cell clones by aqueous humor

尽管htlv -1感染的t细胞中存在炎症因子的组成性过表达，但大多数细胞因子基因在眼部新鲜浸润细胞中不表达，提示细胞因子基因表达受到抑制。为了了解胡的病理生理，我们对房水（AH）中的抑制因子及其抑制机制进行了表征。AH剂量依赖性地抑制htlv -l感染的t细胞克隆和细胞系Sez产生ifn - γ和IL-6。凝胶过滤柱分离AH后发现4个抑制活性峰，明显对应了a-MSH、VIP、MIF和tgf - β的分子质量。然而，AH的主要免疫抑制因子tgf - β 2没有表现出任何抑制活性。我们使用NOKl和NOK3抗fasl单抗，通过ELISA证实了AH中存在sFasL，并发现NOK2单抗可以中和这种抑制。交联Fas选择性抑制ifn - γ mRNA表达。在瞬时荧光素酶实验中，his标记的sFasL显示出剂量依赖性的ifn - γ产生和启动子活性抑制。浓度低至10 ~ l00pg/ml时，抑制作用明显。对ifn - γ启动子的缺失和突变分析发现了一个对fas介导的抑制有24个核苷酸响应的元件，该元件包含YYl和B-box的结合基序。该元素中E-box的突变废除了这种抑制。Fas的一种接头蛋白Daxx的显性阴性表达消除了抑制作用。结果表明，sFasL是眼部细胞因子产生的抑制因子之一，对细胞因子介导的组织损伤具有保护作用。他们还表明sFasL与FasL的膜结合功能不同。

In spite of constitutive overexpression of inflammatory cytokines in HTLV-1-infected T-cells, most cytokine genes are not expressed in the fresh infiltrating cells in the eye, suggesting a suppression of cytokine gene expression. To understand pathophysiology of HU, we characterized the suppressive factors in the aqueous humor (AH) and the mechanisms of suppression. AH dose-dependently suppressed the production of IFN-gamma and IL-6 by HTLV-l-infected T-cell clones and a cell line, Sez. Fractionation of AH by gel filtration column revealed four peaks of suppressive activity, which apparently corresponded molecular masses of a-MSH, VIP, MIF and TGF-beta. However, TGF-beta2, a major immunosuppressive factor in AH, did not show any suppressive activity. We demonstrated sFasL in AH by ELISA using NOKl and NOK3 anti-FasL mAbs and revealed neutralization of the suppression by NOK2 mAb. Crosslinking Fas selectively repressed the IFN-gamma mRNA expression. His-tagged sFasL showed dose-dependent suppression of IFN-gamma production and promoter activity in transient Luciferase assay. Suppression was evident at the concentration as low as 10 to l00pg/ml. Deletion and mutation analyses of IFN-gamma promoter identified a responsive element of 24 nucleotides to the Fas-mediated suppression, which contained binding motifs of YYl and an B-box. Mutation of the E-box in this element abrogated the suppression. Suppression was abolished by expression of dominant negative form of Daxx, an adaptor protein of Fas. The results indicated that sFasL is one of the suppressive factors of cytokine production in the eye, which would play a protective role against cytokine-mediated tissue damages. They also suggested a distinct function of sFasL from the membrane-bound from of FasL.