Modulation of IL-1β, IL-6 and TNF-α Secretion and mRNA Expression by the Trichothecene Vomitoxin in the RAW 264.7 Murine Macrophage Cell Line

Oral exposure of mice to vomitoxin (VT) has been previously shown to enhance gene expression of several cytokines associated with macrophage activation. Here, the effects of exposure to VT in vitro on cytokine secretion and mRNA expression were determined in the murine macrophage cell line RAW 264.7. Enzyme-linked immunosorbent assay (ELISA) of supernatants revealed that significant increases in secreted tumour necrosis factor alpha (TNF-α) were observed 2 days after exposure to VT at 100 ng/ml and 250 ng/ml, both with and without lipopolysaccharide (LPS) activation. While VT did not affect IL-6 secretion in the absence of LPS, significantly increased IL-6 production was observed in culture supernatants after 1, 2 and 5 days of exposure to VT at 250 ng/ml in the presence of LPS. Soluble IL-1β was not detected in control or VT-treated cell cultures with or without LPS activation. Immunochemical staining of intracellular cytokines in conjunction with flow cytometric analysis was used to detect the effects of VT on the percentage of positive cells and output per cell. The percentage of cells that produced intracellular TNF-α were significantly increased at 100 and 250 ng/ml VT with and without LPS whereas increased IL-6 output per cell was observed at 100 and 250 ng/ml VT with LPS. To assess the effects of VT on cytokine mRNA expression, RAW 264.7 cells were analysed semi-quantitatively using reverse transcription-polymerase chain reaction(RT-PCR) in conjunction with Southern hybridization analysis. Elevated TNF-α mRNA was observed at 100 and 250 ng VT/ml at 6 and 24 hr in the absence of LPS. With the addition of LPS, superinduction of TNF-α was not observed in the presence of VT. Increased IL-1β and IL-6 mRNAs were observed at 100 and 250 ng VT/ml at 24 hr in the presence of LPS. These results demonstrated that VT could superinduce both cytokine secretion and mRNA levels in macrophage cultures.