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Influence of HFE variants and cellular iron on monocyte chemoattractant protein-1.

Mitchell RM, Lee SY, Randazzo WT, Simmons Z, Connor JR - J Neuroinflammation (2009)

Bottom Line: The effects of the variants as well as the resulting cellular iron dyshomeostasis potentially impact a number of disease-associated pathways.We further examined the relationship between iron and MCP-1 and found MCP-1 secretion tightly associated with intracellular iron status.Moreover, HFE genotype was a factor in the effect of minocycline, a multifaceted antibiotic used in treating a number of neurologic conditions associated with inflammation, on MCP-1 secretion.

View Article: PubMed Central - HTML - PubMed

Affiliation: George M Leader Family Laboratory, Department of Neurosurgery, Pennsylvania State University College of Medicine/Milton S Hershey Medical Center, Hershey, PA 17033, USA. rmm311@psu.edu

ABSTRACT

Background: Polymorphisms in the MHC class 1-like gene known as HFE have been proposed as genetic modifiers of neurodegenerative diseases that include neuroinflammation as part of the disease process. Variants of HFE are relatively common in the general population and are most commonly associated with iron overload, but can promote subclinical cellular iron loading even in the absence of clinically identified disease. The effects of the variants as well as the resulting cellular iron dyshomeostasis potentially impact a number of disease-associated pathways. We tested the hypothesis that the two most common HFE variants, H63D and C282Y, would affect cellular secretion of cytokines and trophic factors.

Methods: We screened a panel of cytokines and trophic factors using a multiplexed immunoassay in human neuroblastoma SH-SY5Y cells expressing different variants of HFE. The influence of cellular iron secretion on the potent chemokine monocyte chemoattractant protein-1 (MCP-1) was assessed using ferric ammonium citrate and the iron chelator, desferroxamine. Additionally, an antioxidant, Trolox, and an anti-inflammatory, minocycline, were tested for their effects on MCP-1 secretion in the presence of HFE variants.

Results: Expression of the HFE variants altered the labile iron pool in SH-SY5Y cells. Of the panel of cytokines and trophic factors analyzed, only the release of MCP-1 was affected by the HFE variants. We further examined the relationship between iron and MCP-1 and found MCP-1 secretion tightly associated with intracellular iron status. A potential direct effect of HFE is considered because, despite having similar levels of intracellular iron, the association between HFE genotype and MCP-1 expression was different for the H63D and C282Y HFE variants. Moreover, HFE genotype was a factor in the effect of minocycline, a multifaceted antibiotic used in treating a number of neurologic conditions associated with inflammation, on MCP-1 secretion.

Conclusion: Our results demonstrate that HFE polymorphisms influence the synthesis and release of MCP-1. The mechanism of action involves cellular iron status but it appears there could be additional influences such as ER stress. Finally, these data demonstrate a pharmacogenetic effect of HFE polymorphisms on the ability of minocycline to inhibit MCP-1 secretion.

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Impact of iron on MCP-1 secretion. Vector-transfected SH-SY5Y cells (A) and SH-SY5Y cells expressing Wt HFE (B), H63D HFE (C), or C282Y HFE (D) were treated with 10 μM, 5 μM, or 1 μM desferroxamine (DFO) or 10 μM, 30 μM, or 90 μM ferric ammonium citrate (FAC) for 48 hours and the secretion of MCP-1 was determined by ELISA and normalized to cellular protein content. n = 4. *, **, and *** indicate p < 0.05, 0.01, and 0.001 compared to control. NS = not significant.
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Figure 5: Impact of iron on MCP-1 secretion. Vector-transfected SH-SY5Y cells (A) and SH-SY5Y cells expressing Wt HFE (B), H63D HFE (C), or C282Y HFE (D) were treated with 10 μM, 5 μM, or 1 μM desferroxamine (DFO) or 10 μM, 30 μM, or 90 μM ferric ammonium citrate (FAC) for 48 hours and the secretion of MCP-1 was determined by ELISA and normalized to cellular protein content. n = 4. *, **, and *** indicate p < 0.05, 0.01, and 0.001 compared to control. NS = not significant.

Mentions: To directly determine the ability of iron to influence MCP-1 secretion from cells, each cell line was treated with non-toxic doses of DFO and FAC to interrogate the effect of cellular iron modulation on MCP-1 release (Figure 5). DFO treatment resulted in dose-dependent decreased MCP-1 release in vector-transfected (maximum 75%, p < 0.001), Wt HFE (maximum 68%, p < 0.001), and H63D HFE cells (maximum 64%, p < 0.001), at 5 μM and 10 μM, but had no effect in C282Y HFE cells at these concentrations. Treatment with 90 μM FAC resulted in significantly more MCP-1 secretion in Wt HFE cells (maximum 76%, p < 0.001), but had no effect at any concentration in other cell lines. Treatment with either DFO or FAC had no significant effects in cells expressing C282Y HFE, at these concentrations. Higher concentrations of DFO and FAC were not examined because of data suggesting toxicity at higher doses of these two agents (data not shown).


Influence of HFE variants and cellular iron on monocyte chemoattractant protein-1.

Mitchell RM, Lee SY, Randazzo WT, Simmons Z, Connor JR - J Neuroinflammation (2009)

Impact of iron on MCP-1 secretion. Vector-transfected SH-SY5Y cells (A) and SH-SY5Y cells expressing Wt HFE (B), H63D HFE (C), or C282Y HFE (D) were treated with 10 μM, 5 μM, or 1 μM desferroxamine (DFO) or 10 μM, 30 μM, or 90 μM ferric ammonium citrate (FAC) for 48 hours and the secretion of MCP-1 was determined by ELISA and normalized to cellular protein content. n = 4. *, **, and *** indicate p < 0.05, 0.01, and 0.001 compared to control. NS = not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2656486&req=5

Figure 5: Impact of iron on MCP-1 secretion. Vector-transfected SH-SY5Y cells (A) and SH-SY5Y cells expressing Wt HFE (B), H63D HFE (C), or C282Y HFE (D) were treated with 10 μM, 5 μM, or 1 μM desferroxamine (DFO) or 10 μM, 30 μM, or 90 μM ferric ammonium citrate (FAC) for 48 hours and the secretion of MCP-1 was determined by ELISA and normalized to cellular protein content. n = 4. *, **, and *** indicate p < 0.05, 0.01, and 0.001 compared to control. NS = not significant.
Mentions: To directly determine the ability of iron to influence MCP-1 secretion from cells, each cell line was treated with non-toxic doses of DFO and FAC to interrogate the effect of cellular iron modulation on MCP-1 release (Figure 5). DFO treatment resulted in dose-dependent decreased MCP-1 release in vector-transfected (maximum 75%, p < 0.001), Wt HFE (maximum 68%, p < 0.001), and H63D HFE cells (maximum 64%, p < 0.001), at 5 μM and 10 μM, but had no effect in C282Y HFE cells at these concentrations. Treatment with 90 μM FAC resulted in significantly more MCP-1 secretion in Wt HFE cells (maximum 76%, p < 0.001), but had no effect at any concentration in other cell lines. Treatment with either DFO or FAC had no significant effects in cells expressing C282Y HFE, at these concentrations. Higher concentrations of DFO and FAC were not examined because of data suggesting toxicity at higher doses of these two agents (data not shown).

Bottom Line: The effects of the variants as well as the resulting cellular iron dyshomeostasis potentially impact a number of disease-associated pathways.We further examined the relationship between iron and MCP-1 and found MCP-1 secretion tightly associated with intracellular iron status.Moreover, HFE genotype was a factor in the effect of minocycline, a multifaceted antibiotic used in treating a number of neurologic conditions associated with inflammation, on MCP-1 secretion.

View Article: PubMed Central - HTML - PubMed

Affiliation: George M Leader Family Laboratory, Department of Neurosurgery, Pennsylvania State University College of Medicine/Milton S Hershey Medical Center, Hershey, PA 17033, USA. rmm311@psu.edu

ABSTRACT

Background: Polymorphisms in the MHC class 1-like gene known as HFE have been proposed as genetic modifiers of neurodegenerative diseases that include neuroinflammation as part of the disease process. Variants of HFE are relatively common in the general population and are most commonly associated with iron overload, but can promote subclinical cellular iron loading even in the absence of clinically identified disease. The effects of the variants as well as the resulting cellular iron dyshomeostasis potentially impact a number of disease-associated pathways. We tested the hypothesis that the two most common HFE variants, H63D and C282Y, would affect cellular secretion of cytokines and trophic factors.

Methods: We screened a panel of cytokines and trophic factors using a multiplexed immunoassay in human neuroblastoma SH-SY5Y cells expressing different variants of HFE. The influence of cellular iron secretion on the potent chemokine monocyte chemoattractant protein-1 (MCP-1) was assessed using ferric ammonium citrate and the iron chelator, desferroxamine. Additionally, an antioxidant, Trolox, and an anti-inflammatory, minocycline, were tested for their effects on MCP-1 secretion in the presence of HFE variants.

Results: Expression of the HFE variants altered the labile iron pool in SH-SY5Y cells. Of the panel of cytokines and trophic factors analyzed, only the release of MCP-1 was affected by the HFE variants. We further examined the relationship between iron and MCP-1 and found MCP-1 secretion tightly associated with intracellular iron status. A potential direct effect of HFE is considered because, despite having similar levels of intracellular iron, the association between HFE genotype and MCP-1 expression was different for the H63D and C282Y HFE variants. Moreover, HFE genotype was a factor in the effect of minocycline, a multifaceted antibiotic used in treating a number of neurologic conditions associated with inflammation, on MCP-1 secretion.

Conclusion: Our results demonstrate that HFE polymorphisms influence the synthesis and release of MCP-1. The mechanism of action involves cellular iron status but it appears there could be additional influences such as ER stress. Finally, these data demonstrate a pharmacogenetic effect of HFE polymorphisms on the ability of minocycline to inhibit MCP-1 secretion.

Show MeSH
Related in: MedlinePlus