Hepcidin expression by human monocytes in response to adhesion and pro-inflammatory cytokines

Abstract

Background: A previous urine proteomic analysis from our laboratory suggested that hepcidin may be a biomarker for lupus nephritis flare. Immunohistochemical staining of kidney biopsies from lupus patients showed that hepcidin was expressed by infiltrating renal leukocytes. Here we investigated whether inflammatory cytokines relevant to the pathogenesis of lupus nephritis and other glomerular diseases regulate hepcidin expression by human monocytes.

Methods: Human CD14+ monocytes were incubated with interferon alpha (IFNα), interferon gamma (IFNγ), interleukin-6 (IL6), interleukin-1 beta (IL1β), monocyte chemotactic factor-1 (MCP1), or tumor necrosis factor alpha (TNFα). Hepcidin expression was examined by real-time PCR and enzyme immunoassay.

Results: Monocyte hepcidin mRNA increased during adherence to the tissue culture wells, reaching a level 150-fold higher than baseline within 12 h of plating. After accounting for the effects of adhesion, monocytes showed time and dose-dependent up-regulation of hepcidin mRNA upon treatment with IFNα or IL6. One hour of incubation with IFNα or IL6 increased hepcidin mRNA 20 and 80-fold, respectively; by 24 h the mRNA remained 5- and 2.4-fold higher than baseline. IL1β, IFNγ, and MCP-1 did not affect monocyte hepcidin expression. TNFα inhibited hepcidin induction by IL6 in monocytes by 44%. After 24 h of treatment with IFNα or IL6, immunoreactive hepcidin production by monocytes increased 3- and 2.6-fold, respectively.

Conclusion: Human monocytes produce hepcidin in response to adhesion and the pro-inflammatory cytokines IFNα and IL6.

General significance: The appearance of hepcidin in the kidneys or urine during glomerular diseases may be from infiltrating monocytes induced to express hepcidin by adherence and exposure to pro-inflammatory cytokines found in the renal milieu.

Copyright © 2010 Elsevier B.V. All rights reserved.

Figures

Figure 1

Hepcidin mRNA Expression in Human Monocytes and HepG2 cells. Four million human monocytes and 2 million HepG2 cells were cultured for the indicated times. A. Hepcidin mRNA expression in human monocytes over time in vitro. RNA expression was analyzed by real-time RT-PCR in duplicate and hepcidin mRNA levels were normalized to GAPDH mRNA. B. Relative hepcidin mRNA expression by human monocytes and HepG2 cells normalized to GAPDH. C. Relative hepcidin mRNA expression by human monocytes and HepG2 cells normalized to cell numbers. Hepcidin mRNA was expressed as fold-change over freshly isolated cells (before plating) for both monocytes and HepG2 cells. Data are presented as mean ± standard error with n= 5 for each experiment.

Figure 2

Hepcidin mRNA expression after cytokine treatment. Human monocytes (A) and HepG2 cells (C) were treated with 50 ng/ml IL6 or 250 IU/ml IFNα for time-course studies. B. Monocyte hepcidin mRNA regulation by IL6 and IFNα after 24 hours of incubation. D. Monocyte hepcidin mRNA dose-response to IFNα after 1 hour of treatment. E. Real time RT-PCR products after 1 hour of IFNα treatment of human monocytes. The hepcidin mRNA levels were normalized to GAPDH mRNA and then expressed as fold-change over non-treated cells at each time point for monocytes. Data are presented as mean ± standard error with n= 5 for each experiment.

Figure 3

TNFα inhibits hepcidin mRNA expression by human monocytes. Fifty ng/ml of TNFα was added to monocytes in the presence or absence of IL6 or IFNα, and total RNA was isolated after 1 hour of incubation for real time RT-PCR. Hepcidin mRNA levels were normalized to GAPDH mRNA and then expressed as fold-change of TNFα-treated over non-TNFα-treated cells in each of the IL6 or IFNα groups. The data are presented as mean ± standard error with n=3 for each experiment. The * indicates a significant difference compared to control (P

Figure 4

HepG2 and monocytes secrete immunoreactive hepcidin. A. Time-dependent appearance of hepcidin in HepG2-conditioned culture medium. HepG2 cells were treated with 50 ng/ml IL6 or 250 iu/ml IFNα for different times and hepcidin was measured by EIA. B. Dose-dependent levels of hepcidin in CD14+ monocyte-conditioned cell culture medium at 24 hours. CD14+ monocytes were cultured with 10–20 ng/ml IL6 or 250–1000 IU/ml IFNα for 24 hours and hepcidin was measured by EIA. Data in A and B are expressed as mean ± SE (n=6).

Figure 5

SELDI-TOF-MS identification of hepcidin isoforms produced by human monocytes. Monocytes were untreated, or treated with 50 ng/ml IL6 or 250 IU/ml IFNα for 24 hours. Monocyte-conditioned medium was analyzed on CM10 protein chips and hepcidin-20 and 25 standards were spiked into the culture medium at the indicated concentration. A. Representative ions at the position of hepcidin-20. B. Representative ions at the position of hepcidin-25. These spectra are representatives of two independent experiments.