Kornbluth R S, Gregory S A, Edgington T S
Department of Immunology, Scripps Clinic, La Jolla, CA 92037.
J Immunol. 1988 Sep 15;141(6):2006-15.
Under endotoxin-free conditions, unstimulated human PBMC do not release TNF-alpha, as measured in a sensitive assay with 51Cr release in 6 h from actinomycin D-treated WEHI 164 cells. IFN-gamma alone at less than or equal to 10,000 U/ml is insufficient to elicit TNF-alpha release. Similarly, the lymphokine-rich supernatant of PBMC stimulated by allogeneic cells is also insufficient to induce TNF-alpha release in a short term assay. However, when PBMC are first primed with IFN-gamma for 48 h and then exposed to lymphokine supernatant for 6 h, effector cells within the PBMC population are triggered to express TNF-alpha-mediated cytotoxicity. All of the measured cytotoxicity is attributable to TNF-alpha because it could be abolished by a specific anti-TNF-alpha neutralizing mAb. Although IFN-gamma serves to prime PBMC in this assay system, it fails to trigger the release of TNF-alpha. Instead, a second lymphokine (provisionally termed "cytotoxicity triggering factor" (CTF) is required to induce TNF-alpha release from IFN-gamma-primed human PBMC. In kinetic studies, IFN-gamma priming was optimal when PBMC were exposed to IFN-gamma (150 U/ml) for 48 h. In contrast to the prolonged interval for priming, CTF need be present for 6 h or less for maximal induction of TNF-alpha-mediated cytotoxicity. In dose-response studies, IFN-gamma priming (48 h) required at least 4 U/ml and was complete with 20 to 100 U/ml. By using fully primed PBMC, the response to CTF followed a sigmoidal dose-response curve, which allowed the quantitation of CTF in half-maximal units. Activated Th lymphocytes constitute one cellular source for CTF. CTF is produced by cloned allorective T3+T4+T8-M1- Th cells after alloantigen stimulation, and also by nylon wool-purified T cells after stimulation with PMA and A23187 calcium ionophore. Unstimulated T cells do not release CTF. In physicochemical studies, CTF activity elutes from Sephadex G-100 as a major discrete peak of Mr 55 kDa and minor peaks of 14 kDa and greater than 150 kDa. On the basis of multiple criteria, CTF is distinguishable from several other cytokines: IFN-gamma, IL-1, IL-2, GM-CSF, MIF, CSF-1, TNF-alpha, and lymphotoxin (TNF-beta). We conclude that, by acting together, IFN-gamma and CTF provide a lymphokine pathway whereby Ag-responsive human Th cells induce the immunologic release of TNF-alpha from effector cells present in PBMC.
在内毒素-free条件下，未刺激的人外周血单个核细胞（PBMC）不会释放肿瘤坏死因子-α（TNF-α），这是通过一种敏感的检测方法来测定的，该方法是在放线菌素D处理的WEHI 164细胞中，于6小时内检测51Cr释放量。单独的γ干扰素（IFN-γ）浓度小于或等于10,000 U/ml时不足以引发TNF-α释放。同样，同种异体细胞刺激的PBMC富含淋巴因子的上清液在短期检测中也不足以诱导TNF-α释放。然而，当PBMC先用IFN-γ预刺激48小时，然后再暴露于淋巴因子上清液6小时时，PBMC群体中的效应细胞会被触发表达TNF-α介导的细胞毒性。所有测得的细胞毒性都归因于TNF-α，因为它可被特异性抗TNF-α中和单克隆抗体消除。尽管在该检测系统中IFN-γ用于预刺激PBMC，但它未能触发TNF-α的释放。相反，需要第二种淋巴因子（暂称为“细胞毒性触发因子”（CTF））来诱导IFN-γ预刺激的人PBMC释放TNF-α。在动力学研究中，当PBMC暴露于IFN-γ（150 U/ml）48小时时，IFN-γ预刺激效果最佳。与预刺激的较长时间间隔相反，CTF只需存在6小时或更短时间就能最大程度地诱导TNF-α介导的细胞毒性。在剂量反应研究中，IFN-γ预刺激（48小时）至少需要4 U/ml，20至100 U/ml时预刺激完成。通过使用完全预刺激的PBMC，对CTF的反应呈S形剂量反应曲线，这使得能够以半数最大单位定量CTF。活化的Th淋巴细胞是CTF的一个细胞来源。CTF是由克隆的同种异体反应性T3 + T4 + T8 - M1 - Th细胞在同种异体抗原刺激后产生的，也是由尼龙毛纯化的T细胞在用佛波酯（PMA）和A23187钙离子载体刺激后产生的。未刺激的T细胞不释放CTF。在物理化学研究中，CTF活性从葡聚糖凝胶G - 100上洗脱下来，表现为一个主要的离散峰，分子量为55 kDa，还有14 kDa和大于150 kDa的次要峰。基于多项标准，CTF可与其他几种细胞因子区分开来：IFN-γ、白细胞介素-1（IL-1）、白细胞介素-2（IL-2）、粒细胞-巨噬细胞集落刺激因子（GM-CSF）、移动抑制因子（MIF）、集落刺激因子-1（CSF-1）、TNF-α和淋巴毒素（TNF-β）。我们得出结论，通过共同作用，IFN-γ和CTF提供了一条淋巴因子途径，通过该途径，抗原反应性人Th细胞诱导PBMC中效应细胞免疫释放TNF-α。
