Andersson U, Adolf G, Dohlsten M, Möller G, Sjögren H O
Department of Immunology, Stockholm University, Arrhenius Laboratories for Natural Sciences, Sweden.
J Immunol Methods. 1989 Oct 24;123(2):233-40. doi: 10.1016/0022-1759(89)90227-5.
Mononuclear cells from human blood were stimulated to tumor necrosis factor alpha (TNF alpha) or beta (TNF beta) production by the T cell mitogens anti-CD3 antibody (OKT3) or staphylococcal enterotoxin A (SEA). The cells were then fixed and subsequently permeabilized in suspension by the detergent saponin in order to enable TNF alpha- or TNF beta-specific antibodies to enter the cells and interact with cytoplasmic TNF in producer cells. A characteristic morphology of the staining pattern of the two cytokines was noted, with a local accumulation in the cytoplasm in a perinuclear position reflecting the presence of TNF alpha or -beta in the Golgi system. TNF alpha-producing cells appeared 2-3 h after activation of the cultures and increased up to 6 h. The majority of these early TNF alpha-producing cells were monocytes as judged by two-color staining and morphology, but a small fraction of CD4- and CD8-positive T cells was found up to 72 h. TNF beta production started later and peaked 18 or 48 h after OKT3 or SEA stimulation, respectively. The number of TNF beta-producing cells was much larger than that of TNF alpha-producing cells, and approximately 90% of them were CD4-positive T cells. The remaining TNF beta production occurred in CD8-positive T cells and in B cells. Almost every second CD4-positive T cell made TNF beta at the peak of the SEA-induced synthesis. The cytotoxic activity found in the supernatants correlated well with the number of TNF-producing cells found in the cultures. Cells from fresh blood or unstimulated cultures showed no or very few TNF-producing cells.
人血中的单核细胞通过T细胞丝裂原抗CD3抗体（OKT3）或葡萄球菌肠毒素A（SEA）刺激产生肿瘤坏死因子α（TNFα）或β（TNFβ）。然后将细胞固定，随后在悬浮液中用去污剂皂角苷使其通透，以便使TNFα或TNFβ特异性抗体进入细胞并与产生细胞中的细胞质TNF相互作用。注意到两种细胞因子染色模式的特征形态，在核周位置的细胞质中有局部积累，反映了高尔基体系统中TNFα或 -β的存在。培养物激活后2 - 3小时出现产生TNFα的细胞，并持续增加至6小时。通过双色染色和形态学判断，这些早期产生TNFα的细胞大多数是单核细胞，但在72小时内发现一小部分CD4和CD8阳性T细胞。TNFβ的产生开始较晚，分别在OKT3或SEA刺激后18小时或48小时达到峰值。产生TNFβ的细胞数量比产生TNFα的细胞数量多得多，其中约90%是CD4阳性T细胞。其余的TNFβ产生发生在CD8阳性T细胞和B细胞中。在SEA诱导合成的峰值时，几乎每第二个CD4阳性T细胞都产生TNFβ。培养上清液中发现的细胞毒性活性与培养物中发现的产生TNF的细胞数量密切相关。来自新鲜血液或未刺激培养物的细胞未显示或仅有极少的产生TNF的细胞。
