The synergistic effect of phytohemagglutinin and interferon-γ on the expression of tumor necrosis factor-α from RAW 264.7 cells

Tumor necrosis factor-α (TNF-α) is a major cytokine of host immune reaction by foreign agents. Phytohemagglutinin (PHA) is a dynamic contributor to mitogenic stimulation and augmentation of host immune defense. Interferon-γ (IFN-γ) induces induction of cytokines in macrophages and lymphocytes. The aim of this study was to examine the synergistic effects of PHA plus low dose IFN-γ on TNF-α mRNA production, cytosolic levels, and secretion in RAW 264.7 cells. The cells were stimulated with PHA or IFN-γ using various concentrations for various times. The effects of PHA on TNF-α expression appeared in dose- and time-dependent manners. The maximum doses of PHA and IFN-γ to produce them were 300 μg/ml PHA and 10 ng/ml IFN-γ. The optimum time of PHA for the TNF-α mRNA production and release were 6 and 7 h after stimulation, respectively, whereas the time of IFN-γ on them was achieved at 3 and 8 h. Although the TNF-α mRNA production, cytosolic levels, and secretion from the cells were slightly detected under 10 μg/ml PHA and 1 ng/ml IFN-γ, the combination of PHA (10 μg/ml) and IFN-γ (1 ng/ml) greatly increased them, indicating the synergistic effect of PHA plus low dose IFN-γ on TNF-α expression.