NF-κB inhibition leads to increased synthesis and secretion of MIF in human CD4+ T cells

To examine the effects of nuclear factor kappa B (NF-kappa B) inhibition on the secretion of macrophage migration inhibitory factor (MIF) in human CD4(+) T cells. Isolated human CD4(+) T cells were cultured for 24 h with pharmacological inhibitors of NF-kappa B including parthenolide, pyrrolidine dithiocarbamate, BAY 11-7082, gliotoxin, oridonin, andrographolide, and NF-kappa B shRNA. MIF concentration was measured by intracellular flow cytometry, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction. The intracellular concentrations O-2(-), H2O2, and glutathione were measured using the oxidation-sensitive fluorescent dyes dihydroethidium, dichlorodihydrofluorescein diacetate, and monochlorobimane, respectively. The amount of phosphorylated c-Jun was measured by Western blotting. Treatment of CD4(+) T cells with NF-kappa B inhibitors significantly increased MIF concentration in culture supernatants, MIF gene expression, and O-2(-) production, and decreased the intracellular concentrations of MIF, H2O2, and glutathione. Treatment with LY294002 (PI3K inhibitor) and SP600125 (JNK inhibitor) suppressed NF-kappa B inhibitor induced MIF mRNA expression and MIF secretion. LY294002 and SP600125 inhibited the parthenolide-induced phosphorylation of c-Jun. Treatment with H2O2 decreased the amount of intracellular MIF protein and increased MIF concentration in the culture supernatant. N-acetylcysteine, an antioxidant precursor of glutathione, inhibited the parthenolide-induced and H2O2-induced secretion of MIE These results indicate that pharmacological inhibition of NF-kappa B causes the release of MIF through de novo synthesis of MIF and the secretion of preformed MIF in CD4(+) T cells through the production of reactive oxygen species. (C) 2009 Elsevier B.V. All rights reserved.