Seiji Maruo

REGULATION OF T CELL-DEPENDENT AND -INDEPENDENT IL-12 PRODUCTION BY THE THREE TH2-TYPE CYTOKINES IL-10, IL-6, AND IL-4

The production of IL‐12 by macrophages/dendritic cells (Mɸ/DC) is mediated either by a T cell‐dependent pathway that is induced primarily by the interaction of CD40 ligand (CD40L) on activated T cells with CD40 on IL‐12‐producing cells or by a T cell‐independent pathway that is induced by bacteria or bacterial products and enhanced by interferon‐γ (IFN‐γ). In this study we investigated the ability of the Th2‐type cytokines interleukin (IL) ‐10, IL‐6, and IL‐4 to modulate IL‐12 production in Mɸ/DC induced through the two pathways. IL‐12 production was induced in Mɸ/DC from normal mice by stimulation with the combination of IFN‐γ plus lipopolysaccharide (LPS) or Staphylococcus aureus Cowan I (a model for the T cell‐independent pathway) or by co‐culture with Chinese hamster ovary (CHO) cells transfected with the CD40L (a model for the T cell‐dependent pathway). The effects of three Th2‐type cytokines on IL‐12 production by Mɸ/DC through the two pathways were examined. IL‐10 inhibited IL‐12 production induced through both pathways, although the inhibitory effect was more potent on the (IFN‐γ + LPS)‐induced pathway. IL‐6 inhibited only (LPS + IFN‐γ)‐induced IL‐12 production. The effect of IL‐4 was particularly noteworthy: this cytokme inhibited (LPS + IFN‐γ)‐induced IL‐12 production, whereas it potentiated the production of IL‐12 induced by CD40L. Regulation of IL‐12 protein production by IL‐10 and IL‐4 was found to correspond to the levels of mRNA accumulation for the p40 and p35 IL‐12 genes, whereas the presence of IL‐6 during stimulation decreased IL‐12 protein production without affecting steady‐state mRNA levels. These results indicate that IL‐12 production in Mɸ/DC induced through a T cell‐dependent or ‐independent pathway is positively or negatively regulated by particular cytokines at various control levels. J. Leukoc. Biol. 61: 80–87; 1997.

ESTABLISHMENT OF AN IL-12-RESPONSIVE T CELL CLONE : ITS CHARACTERIZATION AND UTILIZATION IN THE QUANTITATION OF IL-12 ACTIVITY

We previously demonstrated that proliferation of terminally differentiated Thl clones depends primarily on an interleukin‐12 (IL‐12)‐paracrme mechanism mediated by their interactions with antigen‐presenting cells (APC) rather than on an IL‐2‐autocrine mechanism. Such a Thl clone (4‐86, C57BL/6 origin) was cultured with recombinant IL‐12 (rIL‐12) in the absence of either antigen or APC. Some cells survived for several passages of culture with only rIL‐12, and by limiting dilution, several clones highly reactive to rIL‐12 alone were obtained. One of these clones, designated 2D6, was found to proliferate strongly in response to less than 1 pg/mL of rIL‐12. This clone exhibited the following surface phenotypes: CD3+, T cell receptor (TCR) αβ+, Vβ11+, NK‐1.1‐; CD4‐ CD8‐; LFA‐1+, ICAM‐1+; and CD28+, CD80+, CD86+, CTLA‐4‐. In accordance with high responsiveness to IL‐12, 2D6 cells were also found to express IL‐12 receptor (IL‐12R) as detected by incubation with rIL‐12 and then staining with anti‐IL‐12 monoclonal antibody (mAb). Stimulation of 2D6 with rIL‐12 resulted in the expression of interferon‐γ (IFN‐γ) and IL‐10 mRNAs and production of these cytokines. The 2D6 clone responded to IL‐2 (vigorously), IL‐7 (moderately), and IL‐4 (mildly) in addition to IL‐12. However, the Ab capture assay using anti‐IL‐12 mAb enabled us to quantify IL‐12‐specific activity contained in a given sample. Thus, this study describes the unique features of the IL‐12‐responsive T cell clone and demonstrates the utilization of this clone in the quantitation of a specific IL‐12 activity. J. Leukoc. Biol. 61: 346–352; 1997.