Elsevier

Immunology Letters

Volume 123, Issue 1, 24 March 2009, Pages 21-30
Immunology Letters

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

https://doi.org/10.1016/j.imlet.2009.01.010Get rights and content

Abstract

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.

Introduction

Macrophage migration inhibitory factor (MIF), identified originally in T cells, is a pro-inflammatory cytokine that is involved in the innate and adaptive immune responses [1]. In addition to T cells, MIF is expressed by a wide spectrum of cells such as B cells, monocytes/macrophages, dendritic cells, eosinophils, neutrophils, and nonimmune cells such as pituicytes, fibroblast-like synoviocytes, endothelial cells, endometrial cells, and renal tubular cells [2]. MIF release by macrophage is stimulated with glucocorticoid and pro-inflammatory cytokines like TNF-α and interferon-γ. [3], [4]. Lipopolysaccharide, staphylococcal toxic-shock syndrome toxin 1 (TSST1), streptococcal pyrogenic exotoxin A, or other pathogens such as malaria, leishmaniasis, cytomegalovirus and influenza virus also induce MIF release from macrophage [1]. T cell activation by specific antigen, mitogens, or anti-CD3 antibodies results in increased MIF mRNA expression and secretion of MIF protein [5].

MIF induced signal transduction is initiated by binding to the extracellular domain of CD74 [6]. CD74 has short intracytoplasmic domain and no motifs for second messenger and it may interact with signal-transducing molecules [6]. A report suggested that CD44 act as an integral member of the CD74 receptor complex leading to MIF signal transduction [7]. MIF activates ERK1/ERK2 signaling, up-regulates TLR4 expression, suppresses p53 activity, inhibits the positive regulatory effects of JUN-activation domain-binding protein 1 (JAB1) on the activity of JNK and AP1, and antagonizes the immunosuppressive effects of glucocorticoid [1], [8]. MIF plays a pivotal role in regulating cell proliferation, inhibiting apoptosis modulating gene expression of inflammatory cytokines, and antibody subclass switching [1], [2], [8]. MIF is implicated in the pathogenesis of a wide range of diseases including infection, cancer, and autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, and psoriasis [1], [9].

The human MIF gene contains DNA-binding sequences for transcription factors such as nuclear factor kappa B (NF-κB), activator protein 1 (AP-1), hypoxia-inducible transcription factor, cAMP response element binding protein, ETS, GATA-1, and stimulatory protein-1 (SP-1) in the immediate 5′-flanking region of MIF [1]. The expression of MIF in human endometrial stromal cells is up-regulated by NF-κB activation in response to human chorionic gonadotropin [10], tumor necrosis factor-alpha (TNF-α) [11], or interleukin 1β [12]. However, little is known about the role of NF-κB in the regulation of MIF gene expression of CD4+ T cells.

NF-κB is a member of Rel family protein, which includes NF-κB1 (p50 and its precursor p105), NF-κB2 (p52 and its precursor p100), c-Rel, RelB, and RelA (p65). These members form homodimers or heterodimers with other proteins [13]. In unstimulated cells, NF-κB exists in the cytoplasm in an inactive form through its physical association with inhibitory IκB proteins. In response to various stimuli, IκB undergoes phosphorylation and subsequent proteolytic degradation, thereby allowing NF-κB to translocate into the nucleus and activate expression of genes associated with cellular proliferation, apoptosis, immune responses, and inflammation [14], [15]. NF-κB has been known to be involved in the pathogenesis of cancer, sepsis, and chronic inflammatory disease such as rheumatoid arthritis, inflammatory bowel disease, asthma, and multiple sclerosis [16]. NF-κB is an attractive target for therapeutic intervention for some inflammatory diseases and cancer [16]. However, NF-κB also participates in cellular functions associated with host defense. Therefore, NF-κB inhibition can cause adverse effects.

Inhibiting the transcriptional activity of NF-κB leads to accumulation of reactive oxygen species (ROS) [17]. While excessive amounts of ROS can be harmful to the cell, they also act as secondary messengers by acting on different levels in the signal transduction pathway [18]. The effects of ROS on signaling pathways are mainly observed in the mitogen-activated protein kinase (MAPK) pathways. ROS promote JNK activation via multiple signaling pathways such as activation of ASK1 and Src kinase and oligomerization of glutathione S-transferase π [19]. NF-κB inhibition can directly activate JNK through down-regulation of Gadd45β, A20 and XIAP (X chromosome-linked inhibitor of apoptosis which mediating the inhibitory activity of NF-κB on the JNK pathway [18]. JNK is involved in many aspects of cellular regulation including cell proliferation, programmed cell death, and gene expression [20].

We hypothesized that inhibition of NF-κB may positively regulate MIF production via accumulation of ROS in CD4+ T cells.

Section snippets

Cells and reagents

Peripheral blood was obtained with a heparin-treated syringe. Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using Ficoll-Hypaque (Pharmacia LKB, Uppsala, Sweden). Anti-CD4 microbeads were used as recommended by the manufacturer (Miltenyi Biotec, Bergisch Gladbach, Germany). In brief, PBMCs were resuspended in 100 μl of MACS buffer [1% bovine serum albumin (BSA), 5 mM EDTA and 0.01% sodium azide]. Anti-CD4 microbeads (10 μl/1 × 107 cells) were added and incubated

Pharmacological NF-κB inhibition induces MIF secretion

NF-κB, AP-1, and nuclear factor of activated T cells (NFAT) are three transcription factors activated in T cells by antigen recognition. To screen for the transcription factors involved in the constitutive production of MIF in CD4+ T cells, we treated CD4+ T cells with pharmacological inhibitors of these transcription factors for 24 h. We used parthenolide and PDTC as NF-κB inhibitors, SP600125 as a JNK inhibitor, curcumin as an AP-1 inhibitor, and cyclosporin A and FK506 as NFAT inhibitors. The

Discussion

Our data show that pharmacological inhibition of NF-κB in CD4+ T cells leads to MIF secretion. There are several ways to specifically inhibit NF-κB activity. We used pharmacological agents to inhibit NF-κB, raising the possible issue of a lack of specificity. However, we believe that this is not a problem in our experiments for the following reasons. First, we used pharmacological NF-κB inhibitors with different structures and mechanisms of action, including parthenolide, PDTC, BAY 11-7082,

Acknowledgments

This work was supported by a grant (R11-2002-098-05001-0) from the Korea Science & Engineering Foundation through the Rheumatism Research Center at the Catholic University of Korea and the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. R01-2008-000-11737-0).

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