Vomitoxin-mediated IL-2, IL-4, and IL-5 superinduction in murine CD4+ T cells stimulated with phorbol ester calcium ionophore: relation to kinetics of proliferation

Abstract

The effects of the trichothecene vomitoxin (VT) on the kinetics of cell proliferation and cytokine production were evaluated in murine CD4+ T cells. The CD4+ cultures were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin to activate protein kinase C and increase cytoplasmic free calcium, respectively, in a range of VT concentrations. Total and viable cell counts at 3, 5, 7, 9, and 11 days revealed delayed or impaired cell proliferation in cultures containing between 50 and 100 ng/ml VT, with complete inhibition being observed at 250 and 500 ng/ml of VT. The VT concentration required to inhibit protein synthesis in a 3-day culture by 50% in this model was 40 ng/ml. When enzyme-linked immunosorbent assay (ELISA) was used to quantitate cytokines, IL-2 levels in control cultures were highest at Day 1 and declined rapidly thereafter, whereas, in VT groups, IL-2 levels were highest at Day 3 and remained elevated up to 11 days. IL-2 levels were elevated by continuous exposure to 100-500 ng/ml of VT with more than 100-fold differences being observed between control and 250 ng/ml VT from Days 5 to 11. When IL-2 levels were expressed on a per viable cell basis, increases were even more marked with as much as 6 log differences being observed between the treatments at 250-500 ng/ml VT and control cultures at Day 7. Supernatant IL-4 and IL-5 levels were also elevated by 100 and 250 ng/ml VT in a dose- and time-dependent fashion compared to control cultures, whereas 500 ng/ml VT was inhibitory. When relative IL mRNA abundance was analyzed during the first 3 days of culture by reverse transcriptase-polymerase chain reaction (RT-PCR) in conjunction with Southern hybridization analysis, IL-2 mRNA levels in Days 1, 2 and 3 in cultures containing 100 and 250 ng/ml VT were greater than corresponding controls. IL-2 mRNA abundance in both control and VT-treated cultures was maximal at Day 1 and decreased rapidly thereafter in controls, whereas much slower rates of IL-2 disappearance were noted in 100 and 250 ng/ml of VT. IL-4 and IL-5 mRNA levels at VT doses of 50 and 100 ng/ml were also elevated compared to controls. Pulsed VT (8 to 48 hr) or cycloheximide (4 to 48 hr) exposure of CD4+ cells enhanced supernatant levels of IL-2 but not IL-4 upon incubation for 24 hr in fresh medium. This effect was not persistent. Taken together, VT enhanced and/or delayed peak IL-2, IL-4, and IL-5 gene expression and secretion in CD4+ T cells stimulated with PMA and ionomycin. Remarkably, cytokine superinduction occurred simultaneously with partial or maximal inhibition of cell proliferation.