Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes.
Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes.
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DOI：
10.1084/jem.170.3.827
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发表时间：
1989-09-01
期刊：
影响因子：
--
通讯作者：
Trinchieri G
中科院分区：
文献类型：
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作者：
Kobayashi M;Fitz L;Ryan M;Hewick RM;Clark SC;Chan S;Loudon R;Sherman F;Perussia B;Trinchieri G
我们从佛波醇二酯诱导的EB病毒转化的人B淋巴母细胞系RPMI 8866的无细胞上清液中鉴定并纯化出一种新型细胞因子，即NK细胞刺激因子（NKSF）。NKSF活性主要与一种70 - kD的阴离子糖蛋白相关。从SDS - PAGE凝胶中分离出的纯化的70 - kD蛋白，经还原后产生两种小分子，分子量分别为40和35 kD，这表明该细胞因子是一种异二聚体。当加入人外周血淋巴细胞（PBL）时，纯化的NKSF制剂可诱导γ - 干扰素产生，并在该活性中与重组白细胞介素 - 2（rIL - 2）协同作用，增强PBL制剂中NK细胞介导的对NK敏感和NK抗性靶细胞系的细胞毒性，并增强T细胞对有丝分裂原凝集素和佛波醇二酯的有丝分裂反应。这三种活性在经过不同纯化步骤（最终纯化了9200倍）后仍然相关，并且纯化的NKSF在0.1 - 10 pM的浓度范围内介导这三种生物学活性。这些数据有力地表明同一分子介导这三种活性，尽管即使在最纯化的NKSF制剂中存在微量污染肽段，我们也不能排除不同生物活性分子被共同纯化的可能性。纯化的NKSF制剂中不存在其他已知细胞因子、NKSF不寻常的分子构象、纯化蛋白的高比活性以及生物学活性谱将NKSF与先前描述的其他细胞因子区分开来。
We have identified and purified a novel cytokine, NK cell stimulatory factor (NKSF), from the cell-free supernatant fluid of the phorbol diester-induced EBV-transformed human B lymphoblastoid cell line RPMI 8866. NKSF activity is mostly associated to a 70-kD anionic glycoprotein. The purified 70-kD protein, isolated from an SDS-PAGE gel, yields upon reduction two small species of molecular masses of 40 and 35 kD, suggesting that this cytokine is a heterodimer. When added to human PBL, purified NKSF preparations induce IFN-gamma production and synergize with rIL-2 in this activity, augment the NK cell-mediated cytotoxicity of PBL preparations against both NK-sensitive and NK- resistant target cell lines, and enhance the mitogenic response of T cells to mitogenic lectins and phorbol diesters. The three activities remain associated through different purification steps resulting in a 9,200-fold purification, and purified NKSF mediates the three biological activities at concentrations in the range of 0.1-10 pM. These data strongly suggest that the same molecule mediates these three activities, although the presence of traces of contaminant peptides even in the most purified NKSF preparations does not allow us to exclude the possibility that distinct biologically active molecules have been co-purified. The absence of other known cytokines in the purified NKSF preparations, the unusual molecular conformation of NKSF, the high specific activity of the purified protein, and the spectrum of biological activities distinguish NKSF from other previously described cytokines.