Overexpression of the hereditary hemochromatosis protein, HFE, in HeLa cells induces and iron-deficient phenotype.
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A transfectant HeLa cell clone expressing HFE under the control of a tetracycline-repressible promoter was generated. HFE expression was fully repressed by the presence of doxycycline, while it was strongly induced by growth in the absence of doxycycline. HFE accumulation was accompanied by a large (approximately 10-fold) decrease in H- and L-ferritin levels, by a approximately 3-4-fold increase in transferrin receptor, and a approximately 2-fold increase in iron regulatory protein activity. These indices of cellular iron deficiency were reversed by iron supplementation complexes. The overexpressed HFE immunoprecipitated together with transferrin receptor, indicating a physical association which is the likely cause for the observed approximately 30% decrease in 55Fe-transferrin incorporation after 18 h incubation. In the HFE-expressing cells the reduction in transferrin-mediated iron incorporation was partially compensated by a approximately 30% increase in non-transferrin iron incorporation from 55Fe-NTA, evident after prolonged, 18 h, incubations. The findings indicate that HFE binding to transferrin receptor reduces cellular iron availability and regulates the balance between transferrin-mediated and non-transferrin-mediated cellular iron incorporation. (+info)
Iron primes hepatic macrophages for NF-kappaB activation in alcoholic liver injury.
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NF-kappaB activation induced by lipopolysaccharide (LPS) in cultured hepatic macrophages (HM) may be abrogated by pretreatment of cells with a lipophilic iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one (L1, deferiprone), suggesting a role for iron in this molecular event [M. Lin, M., R. A. Rippe, O. Niemela, G. Brittenham, and H. Tsukamoto, Am. J. Physiol. 272 (Gastrointest. Liver Physiol. 35): G1355-G1364, 1997]. To ascertain the relevance in vivo of this hypothesis, HM from an experimental model of alcoholic liver injury were examined for the relationship between nuclear factor (NF)-kappaB activation and iron storage. HM showed a significant increase in nonheme iron concentration (+70%), accompanied by enhanced generation of electron paramagnetic resonance-detected radicals (+200%), NF-kappaB activation (+100%), and tumor necrosis factor-alpha (+150%) and macrophage inflammatory protein-1 (+280%) mRNA induction. Treatment of the cells ex vivo with L1 normalized all these parameters. HM content of ferritin protein, ferritin L chain mRNA, and hemeoxygenase-1 mRNA and splenic content of nonheme iron were increased, suggesting enhanced heme turnover as a cause of the increased iron storage and NF-kappaB activation. To test this possibility, increased iron content in HM was reproduced in vitro by phagocytosis of heat-treated red blood cells. Treatment caused a 40% increase in nonheme iron concentration and accentuated LPS-induced NF-kappaB activation twofold. Both effects could be abolished by pretreatment of cells with zinc protoporphyrin, a hemeoxygenase inhibitor. To extend this observation, animals were splenectomized before 9-wk alcohol feeding. Splenectomy resulted in further increments in HM nonheme iron storage (+60%) and NF-kappaB activation (+90%) and mononuclear cell infiltration (+450%), particularly around the iron-loaded HM in alcohol-fed animals. These results support the pivotal role of heme-derived iron in priming HM for NF-kappaB activation and expression of proinflammatory genes in alcoholic liver injury. (+info)
Interactions and aggregation of apoferritin molecules in solution: effects of added electrolytes.
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We have studied the structure of the protein species and the protein-protein interactions in solutions containing two apoferritin molecular forms, monomers and dimers, in the presence of Na(+) and Cd(2+) ions. We used chromatographic, and static and dynamic light scattering techniques, and atomic force microscopy (AFM). Size-exclusion chromatography was used to isolate these two protein fractions. The sizes and shapes of the monomers and dimers were determined by dynamic light scattering and AFM. Although the monomer is an apparent sphere with a diameter corresponding to previous x-ray crystallography determinations, the dimer shape corresponds to two, bound monomer spheres. Static light scattering was applied to characterize the interactions between solute molecules of monomers and dimers in terms of the second osmotic virial coefficients. The results for the monomers indicate that Na(+) ions cause strong intermolecular repulsion even at concentrations higher than 0.15 M, contrary to the predictions of the commonly applied Derjaguin-Landau-Verwey-Overbeek theory. We argue that the reason for such behavior is hydration force due to the formation of a water shell around the protein molecules with the help of the sodium ions. The addition of even small amounts of Cd(2+) changes the repulsive interactions to attractive but does not lead to oligomer formation, at least at the protein concentrations used. Thus, the two ions provide examples of strong specificity of their interactions with the protein molecules. In solutions of the apoferritin dimer, the molecules attract even in the presence of Na(+) only, indicating a change in the surface of the apoferritin molecule. In view of the strong repulsion between the monomers, this indicates that the dimers and higher oligomers form only after partial denaturation of some of the apoferritin monomers. These observations suggest that aggregation and self-assembly of protein molecules or molecular subunits may be driven by forces other than those responsible for crystallization and other phase transitions in the protein solution. (+info)
The role of lactoferrin in the bactericidal function of polymorphonuclear leucocytes.
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Rabbit polymorphonuclear leucocytes contain the iron-binding protein lactoferrin, and can rapidly phagocytose and destroy Pseudomonas aeruginosa. The lactoferrin normally has a large unsaturated iron-binding capacity. If the cells are exposed to a ferritin-antibody complex, large amounts of this are phagocytosed and appear in the cytoplasmic granules and phagosomes. This leads to saturation of the cellular iron-binding protein with Fe. In these circumstances, the bactericidal power of the cells is greatly reduced with the result that some phagocytosed bacteria survive and eventually grow and destroy the cells. An apoferritin-antibody complex used as a control is also phagocytosed but has no effect on the bactericidal power of the cell. The results support the view that lactoferrin plays an essential role in the bactericidal power of the cell. (+info)
Overexpression of wild type and mutated human ferritin H-chain in HeLa cells: in vivo role of ferritin ferroxidase activity.
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Transfectant HeLa cells were generated that expressed human ferritin H-chain wild type and an H-chain mutant with inactivated ferroxidase activity under the control of the tetracycline-responsive promoter (Tet-off). The clones accumulated exogenous ferritins up to levels 14-16-fold over background, half of which were as H-chain homopolymers. This had no evident effect in the mutant ferritin clone, whereas it induced an iron-deficient phenotype in the H-ferritin wild type clone, manifested by approximately 5-fold increase of IRPs activity, approximately 2.5-fold increase of transferrin receptor, approximately 1.8-fold increase in iron-transferrin iron uptake, and approximately 50% reduction of labile iron pool. Overexpression of the H-ferritin, but not of the mutant ferritin, strongly reduced cell growth and increased resistance to H(2)O(2) toxicity, effects that were reverted by prolonged incubation in iron-supplemented medium. The results show that in HeLa cells H-ferritin regulates the metabolic iron pool with a mechanism dependent on the functionality of the ferroxidase centers, and this affects, in opposite directions, cellular growth and resistance to oxidative damage. This, and the finding that also in vivo H-chain homopolymers are much less efficient than the H/L heteropolymers in taking up iron, indicate that functional activity of H-ferritin in HeLa cells is that predicted from the in vitro data. (+info)
Hydrogen ion interactions of horse spleen ferritin and apoferritin.
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The interactions of horse spleen ferritin and its derivative apoferritin with H+ ions were studied by potentiometric and spectrophotometric titration; to aid in data analysis, heats of ionization over a limited pH range and amide content were also determined. Per apoferritin subunit, all tyrosine and cysteine side chains, two of the nine lysine side chains and at least three of the six histidine side chains were found not to titrate; a preliminary but self-consistent analysis of the titration data is proposed. The titration curve of ferritin was identical with that of apoferritin in the pH range 5.5 to 3. In addition, under the conditions used, the reactivities of ferritin histidines to bromoacetate and of ferritin lysines to formaldehyde were identical with those in apoferritin. Above pH 8, a time-dependent titration of the ferritin core occurs which prevents comparison of the titration curves of the two proteins in this region. However, in the pH regions 5.5 to 7.5, two extra groups per subunit titrate reversibly in ferritin relative to apoferritin. Moreover, although the isoionic points of ferritin and apoferritin are identical in water, the isoionic point of ferritin is 0.5 pH unit lower than that of apoferritin in 0.16 to 1 M KCl. The different effects of KCl and NaCl on the two proteins indicate the presence of cation binding sites in ferritin that are absent in apoferritin and possibly also the presence of anion binding sites in apoferritin that are occupied in ferritin by anions of the core. The difference between the isoionic points of the two proteins in KCl has been interpreted to indicate the presence of approximately 2 phosphate residues per ferritin subunit which serve as cation binding sites and which are negatively charged at the isoionic point in KCl. These phosphates may also represent the additional residues that titrate in ferritin between pH 5.5 and 7.5, or may interact with positively charged residues on the inner surface of the ferritin shell, or both. (+info)
Expression and characterization of the chemokine receptors CCR2 and CCR5 in mice.
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The chemokine receptors CCR2 and CCR5 play important roles in the recruitment of monocytes/macrophages and T cells. To better understand the role of both receptors in murine models of inflammatory diseases and to recognize potential problems when correlating these data to humans, we have generated mAbs against murine CCR2 and CCR5. In mice CCR2 is homogeneously expressed on monocytes and on 2--15% of T cells, closely resembling the expression pattern in humans. In contrast to humans, murine NK cells are highly CCR5 positive. In addition, CCR5 is expressed on 3--10% of CD4 and 10--40% of CD8-positive T cells and is weakly detectable on monocytes. Using a model of immune complex nephritis, we examined the effects of inflammation on chemokine receptor expression and found a 10-fold enrichment of CCR5(+) and CCR2(+) T cells in the inflamed kidneys. The activity of various chemokines and the antagonistic properties of the mAbs were measured by ligand-induced internalization of CCR2 and CCR5 on primary leukocytes. The Ab MC-21 (anti-CCR2) reduced the activity of murine monocyte chemotactic protein 1 by 95%, whereas the Ab MC-68 (anti-CCR5) blocked over 99% of the macrophage-inflammatory protein 1alpha and RANTES activity. MC-21 and MC-68 efficiently blocked the ligand binding to CCR2 and CCR5 with an IC(50) of 0.09 and 0.6--1.0 microg/ml, respectively. In good correlation to these in vitro data, MC-21 almost completely prevented the influx of monocytes in thioglycollate-induced peritonitis. Therefore, both Abs appear as useful reagents to further study the role of CCR2 and CCR5 in murine disease models. (+info)
Chemokine and chemokine receptor expression during initiation and resolution of immune complex glomerulonephritis.
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Chemokines participate in leukocyte infiltration, which plays a major role in glomerular injury during immune complex glomerulonephritis (IC-GN). Because target cell expression of chemokine receptors (CCR) is thought to mediate leukocyte migration, the expression pattern of chemokines and CCR in a model of IC-GN was examined. The transient course and predominant glomerular pathology of this model allows the examination of both the induction and resolution phases of IC-GN. GN was induced in mice by daily apoferritin injection for 2 wk. Urine samples and kidneys were obtained at 1, 2, and 4 wk. Albuminuria was noted at 2 wk, but resolved after 4 wk. This was associated with glomerular IC deposits and mesangial proliferation. Glomerular macrophage infiltration was prominent at 1 and 2 wk, which resolved at 4 wk. Expression of monocyte chemoattractant protein-1 (MCP-1) and RANTES mRNA was upregulated at week 1 and decreased to control levels at weeks 2 and 4. The expression was localized to glomeruli by in situ hybridization and immunohistochemistry. The mRNA of CCR1, CCR2, and CCR5 but not CCR3 or CCR4 were upregulated at week 1 and decreased at weeks 2 and 4. Expression of CCR5 was located to the glomerulus by in situ hybridization and quantitative reverse transcription-PCR of isolated glomeruli. In summary, in a model of transient IC-GN, MCP-1 and RANTES and their receptors CCR1, CCR2, and CCR5 are expressed early and are already downregulated at the peak of proteinuria and leukocyte infiltration. Resolution of glomerulonephritis is associated with a return to baseline of chemokine and CCR expression. Therefore, it is concluded that glomerular MCP-1 and RANTES production directs circulating leukocytes that express CCR1, CCR2, and CCR5 into the glomerulus. After initiating GN, MCP-1 and RANTES and their receptors are readily downregulated. (+info)