Biochimica et Biophysica Acta (BBA) - General Subjects
Hepcidin expression by human monocytes in response to adhesion and pro-inflammatory cytokines
Introduction
Hepcidin was initially identified as an antimicrobial peptide in ultrafiltrates of human blood [1] and urine [2], although it is now better known for its role in iron homeostasis. Mature hepcidin is a cysteine-rich 25 amino acid cationic peptide with four intramolecular disulfide bonds. It is predominantly expressed by the liver, although various tissues can express hepcidin at low levels, such as heart, brain and monocytes [3], [4]. Hepcidin binds to ferroportin on cell surfaces, causing its internalization and subsequent degradation [5], [6]. The loss of ferroportin inhibits cellular iron efflux from macrophages and hepatocytes, and decreases intestinal iron absorption, leading to a fall in plasma iron.
Hepcidin expression is increased by iron loading and inflammation, whereas iron deficiency, blood loss, erythropoiesis and hypoxia reduce hepcidin expression [7], [8], [9], [10]. Although the molecular mechanisms of hepcidin regulation are complex, BMP-SMAD signaling appears to have a central regulatory role [7], [8], [9], [10]. Recently endoplasmic reticulum (ER) stress was shown to transcriptionally activate the hepcidin gene promoter through the cyclic AMP response element-binding protein H (CREBH) [11]. Inflammatory cytokines such as IL6 and LPS activate hepcidin transcription through janus kinase and signal transducers and activators of transcription (JAK/STAT) signaling in hepatoma and monocyte cell lines [7], [12], however this pathway also requires BMP-SMAD [12]. Interferon-gamma (IFNγ) alone is a poor activator of hepcidin expression, but acts synergistically with mycobacteria to induce hepcidin in mouse macrophages [13]. Excluding IL6 [14], [15] and LPS [16], [17] cytokine regulation of hepcidin has not been fully investigated, especially in primary human monocytes.
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that affects multiple organs, including the kidneys in 40–60% of patients. In SLE, various cytokines play important roles in recruiting monocytes, T-lymphocytes and neutrophils to mediate local tissue injury [18], [19]. Type I interferons (IFNα and IFNβ), IL6, and TNFα have all been implicated in the pathogenesis of lupus and lupus nephritis [20], [21], [22]. IFNα can modulate immune responses by facilitating dendritic cell maturation from monocytes, promoting long-term antibody and chemokine production, enhancing responses to IL6 and IFNγ, and suppressing responses to TNFα. IL6 plays a critical role in B cell hyperactivity and mesangial cell proliferation. The role of TNFα in LN is complex. Some studies suggest TNFα helps maintain tolerance in SLE, delaying disease appearance [23]. However after SLE is initiated, TNFα may be proinflammatory and TNFα expression is especially associated with LN [23], [24], [25]. All of these cellular and systemic effects of IFNα, TNFα, and IL6 can mediate tissue damage in the kidneys of SLE patients.
A urine proteomic evaluation of lupus nephritis from our laboratory demonstrated that hepcidin may be a biomarker for SLE renal flare [26]. The urine levels of hepcidin-25 and hepcidin-20 changed according to lupus nephritis activity, and leukocytes (probably monocytes) infiltrating the renal interstitium of patients with SLE and active nephritis expressed hepcidin. These findings suggested that hepcidin from infiltrating renal monocytes may contribute to urinary hepcidin expression in lupus nephritis. A limited amount of work has been done on hepcidin regulation in monocytes and macrophages. Most studies have used the human monocyte cell line THP-1, or murine peritoneal macrophages, and the stimuli were IL6 or infection [13], [27], [28]. Work from Weiss et al. demonstrated that both IL6 and LPS induced hepcidin in a human monocyte cell line, but other cytokines were not explored [4]. The present study was undertaken to characterize the regulation of hepcidin expression in primary human monocytes by inflammatory cytokines relevant to lupus and lupus nephritis (LN).
Section snippets
Human monocyte isolation and culture
Peripheral blood mononuclear cells (PBMC) were isolated from American Red Cross source leukocytes (product code 20301) using Cellgro Lymphocyte Separation Medium (Mediatech Herndon, VA). These cells were from multiple donors and replicate experiments were done with cells from different donors. CD14+ monocytes were then further purified by MACS CD14 MicroBeads (Miltenyi Biotech, Auburn, CA). Approximately 4 million CD14+ monocytes were plated in each well of 12-well plates in RPMI 1640 containing
Monocyte hepcidin mRNA increases during adhesion
After immunohistochemistry indicated that renal interstitial leukocytes in the kidneys of patients with LN produced hepcidin [26], we postulated that cytokines present in the kidney during LN could regulate monocyte hepcidin expression. To test this hypothesis, hepcidin mRNA was first measured in monocytes derived from PBMC after they were allowed to adhere to tissue culture wells for different lengths of time. As shown in Fig. 1A, CD14+ monocyte hepcidin mRNA began to increase after 1 h in
Acknowledgments
We thank Nathan Harris (Molecular Sensing, Montara, CA) for his help in SELDI analysis. This research work is supported by NIH/NIDDK research grant R21 DK0773311.
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