Enzyme-linked immunospot assays provide a sensitive tool for detection of cytokine secretion by monocytes
Enzyme-linked immunospot assays provide a sensitive tool for detection of cytokine secretion by monocytes
复制标题
DOI：
10.1128/cdli.8.6.1248-1257.2001
复制
发表时间：
2001-11-01
期刊：
影响因子：
--
通讯作者：
Link, H
中科院分区：
文献类型：
--
作者：
Kouwenhoven, M;Özenci, V;Link, H
血液单核细胞以及组织分化的巨噬细胞在控制免疫反应中起关键作用。单核细胞通过分泌细胞因子调节免疫应答的程度、性质和持续时间。白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）、IL-10和IL-12是特别令人感兴趣的，因为IL-12使免疫应答向Th 1型转变，促进产生例如，TNF-α和IL-6，而IL-10抵消Th 1应答并促进Th 2相关细胞因子如IL-4的产生。这四种细胞因子的严格调节保持平衡，并决定Th 1或Th 2在免疫反应中占主导地位。酶联免疫斑点法（ELISPOT）是细胞因子研究中最敏感和特异的方法之一。它们允许离体鉴定主动分泌细胞因子的单个细胞。在本研究中，我们从健康受试者的血液中制备单核细胞，并采用ELISPOT测定法来确定检测和计数分泌IL-6、TNF-α、IL-10和IL-12的单核细胞的最佳条件。单核细胞孵育的最佳时间为24小时，最佳单核细胞数量（以细胞/孔计）为IL-6为2，000，TNF-α为1，000，IL-10为50，000，IL-12分泌单核细胞计数为100，000。在健康受试者中，10% +/- 5%的单核细胞分泌IL-6，12% +/- 12%分泌TNF-α，0.1% +/- 0.1%分泌IL-10，0.2% +/- 0.3%分泌IL-12（数值为平均值标准差）。总之，ELISPOT分析构成了一种有价值的工具，用于计数分泌IL-6、TNF-α、IL-10和IL-12的单核细胞，并可能计数分泌其他细胞因子和蛋白质的单核细胞。
Blood monocytes as well as tissue-differentiated macrophages play a pivotal role in controlling immune reactions. Monocytes regulate the extent, nature, and duration of immune responses by secretion of cytokines. Interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), IL-10, and IL-12 are of particular interest, since IL-12 shifts the immune response towards a Th1 type, facilitating the production of, e.g., TNF-alpha and IL-6, while IL-10 counteracts Th1 responses and promotes the production of Th2-related cytokines such as IL-4. A tight regulation of these four cytokines keeps the balance and decides whether Th1 or Th2 will predominate in immune reactions. Enzyme-linked immunospot (ELISPOT) assays are among the most-sensitive and -specific methods available for cytokine research. They permit ex vivo identification of individual cells actively secreting cytokines. In the present study we prepared monocytes from healthy subjects' blood and adapted ELISPOT assays to define optimal conditions to detect and enumerate monocytes secreting IL-6, TNF-alpha, IL-10, and IL-12. The optimal time for monocyte incubation was 24 h, and optimal monocyte numbers (in cells per well) were 2,000 for IL-6, 1,000 for TNF-alpha, 50,000 for IL-10, and 100,000 for enumeration of IL-12 secreting monocytes. Among healthy subjects, 10% +/- 5% of the monocytes secreted IL-6, 12% +/- 12% secreted TNF-alpha, 0.1% +/- 0.1% secreted IL-10, and 0.2% +/- 0.3% secreted IL-12 (values are means standard deviations). In conclusion, ELISPOT assays constitute a valuable tool to enumerate monocytes secreting IL-6, TNF-alpha, IL-10, and IL-12 and probably to enumerate monocytes secreting other cytokines and proteins.