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Iron infusion activates synovium and induced joint inflammation in experimental animals and causes the flaring up of arthritis in patients with rheumatoid arthritis (RA). Marked iron deposition in RA synovia has been reported over the past 30 years and has also recently been demonstrated by quantitative photometric assessment and is correlated with exudative and proliferative histological features.1 It has been reported that the amount of iron deposition in RA synovial tissue is correlated with disease activity and severity. Iron has an important role in RA synovitis through the formation of radical oxygen species, and the enhancement of collagen synthesis and synovial fibroblast proliferation2 possibly owing to down regulation of prostaglandin E2 (PGE2) production.3
Synovial fibroblasts produce a number of inflammatory mediators including cytokines such as interleukin (IL)1, IL6, IL8, fibroblast growth factor, vascular endothelial growth factor, tumour necrosis factor, and granulocyte macrophage colony stimulating factor (GM-CSF). GM-CSF produces the progenitor cells of macrophage lineage stem cells and stimulates mature granulocytes and macrophages. GM-CSF is produced by T cells, macrophages, and fibroblasts and has been found in synovial fluid and tissue from patients with RA. GM-CSF has an important role in type II collagen induced arthritis in rats and in the acute methylated bovine serum albumin induced murine arthritis model. A protective effect against collagen induced arthritis was seen in GM-CSF knockout mice. Using those mice, it has been recently shown that GM-CSF plays a part in the IL1 induced arthritis that follows methylated bovine serum albumin injection.4 In this study we showed that iron enhanced GM-CSF but did not enhance IL6 or IL8 production by human synovial fibroblasts on stimulation with IL1β in vitro.
Synovial tissues were obtained from 20 patients, 11 with RA and nine with osteoarthritis. The synovial fibroblasts were isolated according to a method described previously.2 The material containing the synovial fibroblasts during the third to seventh passage was used in the experiments. Synovial fibroblasts were added at a concentration of 1×104 cells/well to each well of a 96 well microtitre plate, and cultured for 18, 24, 48, 72, 96, and 120 hours with or without recombinant human IL1β. The cytokines in the culture supernatant were measured with a commercially available enzyme linked immunosorbent assay (ELISA) kit (Amersham Life Science, UK). Ferric citrate was used as an iron salt or sodium citrate as a control. The differences in cytokine production by synovial fibroblasts cultured with ferric citrate compared with sodium citrate or medium alone were analysed by paired Student’s t test. A level of p<0.05 was accepted as significant.
To investigate the effects of iron on GM-CSF production by synovial fibroblasts, three different concentrations of ferric or sodium citrate (0.01, 0.1, and 1 mmol/l at final concentration) were added to synovial fibroblast 96 hour culture with three different concentrations of IL1β (1, 10, and 100 ng/ml at final concentration). Ferric citrate (1 mmol/l) but not sodium citrate significantly enhanced GM-CSF production by synovial fibroblasts on stimulation with 1 ng/ml of IL1β (fig 1A). Ferric citrate (0.1 mmol/l) enhanced GM-CSF production by synovial fibroblasts on stimulation with 10 ng/ml of IL1β (fig 1B). Also, concentrations of 0.01 or 0.1 mmol/l ferric citrate enhanced production of GM-CSF by synovial fibroblasts on stimulation with 100 ng/ml of IL1β (fig 1C). However, ferric citrate at any concentration tested did not enhance IL6 or IL8 production by synovial fibroblasts on stimulation with any concentration of IL1β including 100 ng/ml (data not shown).
To test whether iron regulates the transcriptional level of GM-CSF production by synovial fibroblasts on stimulation with IL1β, GM-CSF, IL6 and IL8 mRNA expression in synovial fibroblasts were examined semiquantitatively by reverse transcriptase-polymerase chain reaction (RT-PCR). The method used for RT-PCR using specific primers for cytokines and glyceraldehyde-3-phosphate dehydrogenase as a control has been described in detail previously.5 The expression of mRNA encoding these cytokines in synovial fibroblasts was undetectable without IL1, and was dose dependent on IL1β reaching a plateau after three hours in culture. Ferric citrate (0.1 mmol/l) significantly enhanced IL1 induced GM-CSF mRNA expression in synovial fibroblasts but not that of IL6 or IL8 (data not shown).
GM-CSF is produced by T cells, macrophages, mast cells, endothelial cells, and fibroblasts in response to specific activating signals. GM-CSF gene expression is controlled by binding of transcription factors such as NF-GMa, NF-GMb, NF-κB, Elf-1, NF-AT/AP1 complex, and Sp1 related complex to their specific promoter regions.6 It is unknown which transcription factors participate in the iron mediated regulation of IL1 induced GM-CSF production by synovial fibroblasts. Dlaska and Weiss recently showed that iron regulates the transcription of inducible nitric oxide synthase (iNOS) of macrophage-like cells stimulated with interferon γ (IFNγ) and/or lipopolysaccharide, and binding of NF-IL6 to its consensus motif within the iNOS promoter was reduced by iron and enhanced by an iron chelator.7 We found that iron enhanced IL1 induced GM-CSF production by synovial fibroblasts. Taken together, the intracellular iron levels might control GM-CSF production induced by IL1 by mechanism(s) similar to the action of iron-responsive element binding proteins on ferritin or transferrin receptor synthesis or iNOS induction.8
We found that the effects of iron on GM-CSF production induced by IL1 were different from those on IL6 or IL8 production. Agro et al reported that PGE2 enhanced IL6 and IL8 but inhibited GM-CSF production by IL1 stimulated synovial fibroblasts.9 Previously, we showed that iron decreased PGE2 production by synovial fibroblasts.3 Iron might enhance GM-CSF production through down regulation of PGE2 production. Yoshida et al found that gold compounds and divalent metal ions inhibited induction of IL6 and IL8 but not production of GM-CSF by IL1 induced synovial fibroblasts through inhibition of NF-κB binding to DNA.10 Thus, there may be different signal transduction pathways among these three cytokines in IL1 stimulated human synovial fibroblasts.
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