Enzyme-linked immunospot assays provide a sensitive tool for detection of cytokine secretion by monocytes

Blood monocytes as well as tissue-differentiated macrophages play a pivotal role in controlling immune reactions. Monocytes regulate the extent, natur e, 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 t ype, facilitating the production of, e.g., TNF-alpha and IL-6, while IL-10 counteracts Th1 responses and promotes the production of Th2-related cytoki nes such as IL-4. A tight regulation of these four cytokines keeps the bala nce and decides whether Th1 or Th2 will predominate in immune reactions. En zyme-linked immunospot (ELISPOT) assays are among the most-sensitive and -s pecific methods available for cytokine research. They permit ex vivo identi fication of individual cells actively secreting cytokines. In the present s tudy we prepared monocytes from healthy subjects' blood and adapted ELISPOT assays to define optimal conditions to detect and enumerate monocytes secr eting IL-6, TNF-alpha, IL-10, and IL-12. The optimal time for monocyte incu bation was 24 h, and optimal monocyte numbers (in cells per well) were 2,00 0 for IL-6, 1,000 for TNF-alpha, 50,000 for IL-10, and 100,000 for enumerat ion 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% sec reted IL-10, and 0.2% +/- 0.3% secreted IL-12 (values are means standard de viations). In conclusion, ELISPOT assays constitute a valuable tool to enum erate monocytes secreting IL-6, TNF-alpha, IL-10, and IL-12 and probably to enumerate monocytes secreting other cytokines and proteins.