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

尽管炎症细胞因子在HTLV-1感染的T细胞中呈结构性过表达，但大多数细胞因子基因在眼内新鲜的浸润性细胞中不表达，这表明细胞因子基因的表达受到抑制。为了了解HU的病理生理学，我们描述了房水中的抑制因素及其抑制机制。AH剂量依赖性地抑制HTLV-L感染的T细胞克隆和细胞株SEZ产生干扰素-γ和IL-6。用凝胶过滤柱层析分离的AH有四个抑制活性峰，分别与α-MSH、VIP、MIF和TGF-β的相对分子质量相对应。然而，作为急性肝炎主要免疫抑制因子的转化生长因子-β2没有表现出任何抑制活性。我们用NOK1和NOK3抗FasL单抗用ELISA法证实了AH中的sFasL，并发现NOK2单抗中和了这种抑制作用。交联型Fas选择性抑制干扰素-γ的mRNA表达。瞬时荧光素酶检测显示，His标记的sFasL对干扰素-γ的产生和启动子活性有剂量依赖性的抑制作用。对干扰素-γ启动子的缺失和突变分析表明，有24个核苷酸对Fas介导的抑制有反应，其中含有YY1和B-box的结合基序。该元件中E-box的突变取消了抑制作用。Fas的接头蛋白Daxx的显性负性形式的表达取消了抑制作用。结果表明，sFasL是眼内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。他们还提出了sFasL有别于FasL的膜结合形式的独特功能。

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.