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-κB) 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-κB including parthenolide, pyrrolidine dithiocarbamate, BAY 11-7082, gliotoxin, oridonin, andrographolide, and NF-κB shRNA. MIF concentration was measured by intracellular flow cytometry, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction. The intracellular concentrations O₂^-, 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-κB inhibitors significantly increased MIF concentration in culture supernatants, MIF gene expression, and O₂^- production, and decreased the intracellular concentrations of MIF, H₂O₂, and glutathione. Treatment with LY294002 (PI3K inhibitor) and SP600125 (JNK inhibitor) suppressed NF-κB 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 MIF. These results indicate that pharmacological inhibition of NF-κB 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.