Bethea J R, Gillespie G Y, Chung I Y, Benveniste E N
Department of Neurology, University of Alabama, Birmingham 35294.
J Neuroimmunol. 1990 Nov;30(1):1-13. doi: 10.1016/0165-5728(90)90047-q.
Human malignant gliomas possess some of the same immune-related functions as astrocytes do. For instance, they are capable of secreting various immunoregulatory molecules and expressing HLA-DR antigens on their surface. The human malignant glioma cell line, D54-MG, was used to investigate the proliferative effects of tumor necrosis factor-alpha (TNF-alpha) and the expression of specific surface receptors for TNF-alpha. Additionally, we were interested in examining whether D54-MG cells are capable of synthesizing and secreting biologically active TNF-alpha. D54-MG cells responded in a mitogenic fashion upon incubation with TNF-alpha for 48 h under serum-free conditions. 125I-labeled TNF-alpha was used in this study to investigate the expression of receptors specific for TNF-alpha on D54-MG cells. Scatchard analysis of our receptor binding data produced curvilinear plots indicating there are two distinct receptor sites for TNF-alpha. From these data, we calculated that there are approximately 3500 high affinity and 24,666 low affinity binding sites per cell. Pretreating these cells with interferon-gamma (IFN-gamma) resulted in a 2-fold increase in the number of high affinity binding sites and a moderate increase in the number of low affinity binding sites, with no appreciable change in binding affinity (Kd) of either site. D54-MG cells were unable to constitutively secrete TNF-alpha; however, upon stimulation, these cells synthesize and secrete biologically active TNF-alpha. Polyclonal antisera reactive with human macrophage-derived TNF-alpha neutralized the cytotoxicity of D54-MG-derived TNF-alpha, demonstrating that the cytotoxic activity was in fact due to TNF-alpha. Our observations indicate that TNF-alpha could act in an autocrine fashion to induce the proliferation of this malignant glioma cell line and that TNF-alpha exerts its effect by binding to specific TNF-alpha receptors whose expression was enhanced by IFN-gamma.
人类恶性胶质瘤具有一些与星形胶质细胞相同的免疫相关功能。例如，它们能够分泌各种免疫调节分子并在其表面表达HLA - DR抗原。使用人类恶性胶质瘤细胞系D54 - MG来研究肿瘤坏死因子 - α（TNF - α）的增殖作用以及TNF - α特异性表面受体的表达。此外，我们还想研究D54 - MG细胞是否能够合成和分泌具有生物活性的TNF - α。在无血清条件下，D54 - MG细胞与TNF - α孵育48小时后以促有丝分裂方式作出反应。本研究使用125I标记的TNF - α来研究D54 - MG细胞上TNF - α特异性受体的表达。对我们的受体结合数据进行Scatchard分析产生了曲线图谱，表明TNF - α存在两个不同的受体位点。根据这些数据，我们计算出每个细胞大约有3500个高亲和力和24666个低亲和力结合位点。用干扰素 - γ（IFN - γ）预处理这些细胞导致高亲和力结合位点数量增加2倍，低亲和力结合位点数量适度增加，而两个位点的结合亲和力（Kd）没有明显变化。D54 - MG细胞不能组成性地分泌TNF - α；然而，在受到刺激后，这些细胞合成并分泌具有生物活性的TNF - α。与人类巨噬细胞衍生的TNF - α反应的多克隆抗血清中和了D54 - MG衍生的TNF - α的细胞毒性，表明细胞毒性活性实际上是由TNF - α引起的。我们的观察结果表明，TNF - α可能以自分泌方式作用于诱导这种恶性胶质瘤细胞系的增殖，并且TNF - α通过与特定的TNF - α受体结合发挥其作用，其表达因IFN - γ而增强。
