Michishita M, Yoshida Y, Uchino H, Nagata K
First Division of Internal Medicine, Faculty of Medicine, Kyoto University, Japan.
J Biol Chem. 1990 May 25;265(15):8751-9.
We examined the characteristics of tumor necrosis factor (TNF) receptors expressed on immature mouse myeloid leukemic cells (M1), M1 cells induced to differentiate into macrophages, and macrophage cells (Mm1 cells) by binding studies with radioiodinated TNF. Scatchard analysis of TNF binding revealed that a single class of high affinity receptor was present and that 750-1,100 receptors were expressed on each immature M1 cell. The number of TNF receptors was increased 1.5-2-fold on differentiated M1 cells and 4-5-fold on Mm1 cells with no change in affinity. The addition of interferon-gamma (IFN-gamma) up-regulated the expression of TNF receptors in differentiated M1 cells and Mm1 cells, while immature M1 cells were insensitive to IFN-gamma. The number of TNF receptors on the differentiated cells was increased 4-5-fold by the treatment with IFN-gamma with no change in the binding constant. The affinity of TNF receptors to human TNF-alpha (Kd = 1.7-2.8 nM) was lower than that to murine TNF-alpha (Kd = 0.2-0.7 nM). The assays for cell growth and [3H]thymidine incorporation suggested that no relation exists between the sensitivity of the cells to TNF-alpha and the number of TNF receptors. Enhancement of TNF-mediated cytotoxicity by the treatment with IFN-gamma did not correlate with increases in the number of TNF receptors. Cytolytic assays using L929 cells demonstrated that the amount of constitutive and lipopolysaccharide (LPS)-induced secretion of TNF-alpha was markedly increased during differentiation. Both the constitutive expression and IFN-gamma-mediated superinduction of TNF receptors, and the constitutive and LPS-induced secretion of TNF-alpha were closely related to the extent of cellular differentiation along the monocytic pathway. The time course of LPS-induced TNF-alpha activity showed a rise-and-decline profile with a peak at 2 h. On the other hand, the time course of the number of cell surface TNF receptors showed a decline-and-rise profile, a mirror image of the TNF-alpha activity time course profile in the supernatant. Anti-TNF-alpha antibody treatment blocked the LPS-induced down-regulation of TNF receptors and increased TNF-alpha mRNA accumulation. We discussed "an autoinhibitory system" in which an internalization of secreted TNF-alpha mediated by its own receptors is involved not only in decreasing TNF-alpha activity in the supernatant but also in reducing TNF-alpha mRNA expression.
我们通过用放射性碘化肿瘤坏死因子（TNF）进行结合研究，检测了未成熟小鼠髓性白血病细胞（M1）、诱导分化为巨噬细胞的M1细胞以及巨噬细胞（Mm1细胞）上表达的TNF受体的特性。TNF结合的Scatchard分析表明存在一类单一的高亲和力受体，每个未成熟M1细胞上表达750 - 1100个受体。分化的M1细胞上TNF受体的数量增加了1.5 - 2倍，Mm1细胞上增加了4 - 5倍，而亲和力没有变化。添加干扰素 - γ（IFN - γ）上调了分化的M1细胞和Mm1细胞中TNF受体的表达，而未成熟M1细胞对IFN - γ不敏感。用IFN - γ处理后，分化细胞上TNF受体的数量增加了4 - 5倍，结合常数没有变化。TNF受体对人TNF - α（Kd = 1.7 - 2.8 nM）的亲和力低于对鼠TNF - α（Kd = 0.2 - 0.7 nM）的亲和力。细胞生长和[³H]胸苷掺入试验表明，细胞对TNF - α的敏感性与TNF受体的数量之间不存在关联。用IFN - γ处理增强TNF介导的细胞毒性与TNF受体数量的增加无关。使用L929细胞的细胞溶解试验表明，在分化过程中，TNF - α的组成性分泌和脂多糖（LPS）诱导的分泌量均显著增加。TNF受体的组成性表达和IFN - γ介导的超诱导，以及TNF - α的组成性分泌和LPS诱导的分泌均与沿单核细胞途径的细胞分化程度密切相关。LPS诱导的TNF - α活性的时间进程呈现出上升 - 下降的曲线，在2小时达到峰值。另一方面，细胞表面TNF受体数量的时间进程呈现出下降 - 上升的曲线，与上清液中TNF - α活性时间进程曲线呈镜像关系。抗TNF - α抗体处理阻断了LPS诱导的TNF受体下调，并增加了TNF - α mRNA的积累。我们讨论了“一种自抑制系统”，其中由其自身受体介导的分泌型TNF - α的内化不仅参与降低上清液中TNF - α的活性，还参与减少TNF - α mRNA的表达。
