NF-kappaB inhibition leads to increased synthesis and secretion of MIF in human CD4+ T cells.
To examine the effects of nuclear factor kappa B (NF-kappaB) inhibition on the secretion of macrophage migration inhibitory factor (MIF) in human CD4(+) T cells. Isolated human CD4(+) T cells were cultured for 24h with pharmacological inhibitors of NF-kappaB including parthenolide, pyrrolidine dithiocarbamate, BAY 11-7082, gliotoxin, oridonin, andrographolide, and NF-kappaB shRNA. MIF concentration was measured by intracellular flow cytometry, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction. The intracellular concentrations O(2)(-), H(2)O(2), 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-kappaB inhibitors significantly increased MIF concentration in culture supernatants, MIF gene expression, and O(2)(-) production, and decreased the intracellular concentrations of MIF, H(2)O(2), and glutathione. Treatment with LY294002 (PI3K inhibitor) and SP600125 (JNK inhibitor) suppressed NF-kappaB inhibitor induced MIF mRNA expression and MIF secretion. LY294002 and SP600125 inhibited the parthenolide-induced phosphorylation of c-Jun. Treatment with H(2)O(2) 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 H(2)O(2)-induced secretion of MIF. These results indicate that pharmacological inhibition of NF-kappaB 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.
The Center for Rheumatic diseases in Kangnam St. Mary's Hospital, and Rheumatism Research Center, College of Medicine, The Catholic University of Korea, Seoul, South Korea.
Mi-La
ML
Cho
Young-Mee
YM
Moon
Yu-Jung
YJ
Heo
Yun-Ju
YJ
Woo
Ji-Hyeon
JH
Ju
Kyung-Su
KS
Park
Sung-Il
SI
Kim
Sung-Hwan
SH
Park
Ho-Youn
HY
Kim
Jun-Ki
JK
Min
D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000276:immunology / Q000378:metabolism; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000096:biosynthesis / Q000378:metabolism*; D008263:Macrophage Migration-Inhibitory Factors / Q000096:biosynthesis / Q000378:metabolism*; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009195:Peroxidase / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000276:immunology / Q000378:metabolism
D004791:Enzyme Inhibitors; D008263:Macrophage Migration-Inhibitory Factors; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D009195:Peroxidase; D019746:Intramolecular Oxidoreductases; C515598:MIF protein, human; D005978:Glutathione