INCREASED IL-1, IL-6 AND TNF-ALPHA SECRETION AND MESSENGER-RNA LEVELSIN WEHI-3 CELLS EXPOSED TO CYCLOPIAZONIC ACID

The effects of the mycotoxin cyclopiazonic acid (CPA) on cytokine secr etion and gene expression were evaluated in the WEHI-3 murine macropha ge cell line. Lipopolysaccharide (LPS)-stimulated and non-stimulated c ells were exposed to various concentrations of CPA and culture superna tants were assessed for interleukin (IL)-1 beta, IL-6 and TNF alpha by ELISA. Without LPS stimulation, only IL-6 was increased by CPA at 500 0 ng/ml after 1, 2 and 3 days. With LPS stimulation, IL-1 beta was ele vated in the presence of 500 and 1000 ng/ml of CPA at 1 day and 500, 1 000 and 5000 ng/ml at 2 days and 3 days. TNF alpha was increased by 10 00 ng/ml CPA at 12 h and by 500, 1000 and 5000 ng/ml CPA at 1-3 days. IL-6 levels were increased in the presence of 100, 500 and 1000 CPA ng /ml at both 12 h and 3 days and in the presence of 100, 500, 1000 and 5000 ng/ml CPA at both 1 day and 2 days. The cytokine effects were fur ther related to proliferation and cell viability using the MTT [3-(4,5 -dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide] assay. Prolife ration was increased relative to controls in the presence of 50-1000 n g/ml of CPA in LPS-stimulated cells and in the presence of 500-1000 ng /ml CPA in unstimulated cells. In contrast, proliferation was markedly inhibited by 5000 ng/ml CPA in both stimulated and unstimulated cells . To relate the effect of CPA on IL secretion to mRNA transcript level s, LPS-stimulated cells were incubated with 1000 ng/ml of CPA for 2, 4 , 8, 12 and 24 h and cytokine mRNA levels were evaluated using RT-PCR in combination with Southern hybridization analysis. In the presence o f LPS only, IL-1 beta and IL-6 mRNA peaked at 8 h and 4 h, respectivel y, and then decreased whereas TNF alpha mRNA was strongly expressed fr om 2-8 h and markedly decreased at 12 h. In the presence of LPS and CP A, however, IL-1 beta and IL-6 mRNA levels gradually increased up to 2 4 h reaching 2.5 and 29-fold higher than controls, respectively. In co ntrast, TNF alpha mRNA levels slowly decreased after 8 h but remained markedly elevated relative to controls. Taken together, these results suggest that CPA can superinduce both secretion and mRNA levels of pro inflammatory cytokines associated with macrophage activation. Cytokine upregulation was not always consistent with proliferative effects.